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Ann Thorac Surg 1999;68:1008-1013
© 1999 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, Washington University School of Medicine, St. Louis, Missouri, USA
b Department of Surgery, Washington University School of Medicine, St. Louis, Missouri, USA
c Department of Pathology, 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-Jewish Hospital Plaza, St. Louis, MO 63110
Presented at the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.
Abstract
Background. We investigated endobronchial transfection of CAT and TGF-ß1 cDNA selectively delivered to the lung graft with or without liposomes.
Methods. Phase I: F344 rats received 130 µg of naked plasmid pCF1-CAT or complexed to liposome GL67 via left main bronchus instillation. Rats were awakened (pCF1-CAT, n = 4; GL67:pCF1-CAT, n = 4) or served as donors in an isogenic transplant (pCF1-CAT, n = 5; GL67:pCF1-CAT, n = 5). ELISA was performed on lungs, hearts, and livers on POD 2. Phase II: BN lungs received TGF-ß1 sense (n = 6); antisense (n = 5); GL67:TGF-ß1 sense (n = 10); or saline solution (n = 10). F344 recipients were sacrificed on POD 5. The arterial pO2 and rejection were assessed. RT-PCR for murine TGF-ß1 was performed.
Results. Phase I: CAT expression was 519 ± 287 pg and 63 ± 68 with pCF1-CAT and 104 ± 67 and 37 ± 45 with GL67:pCF1-CAT, respectively, in the non-transplant and in the transplant setting. No protein was detected in the hearts, livers, and in the native lung of the recipients. Phase II: RT-PCR confirmed murine TGF-ß1 transfection. pO2 was 362.7 ± 110.2 (mean mmHg ± SD) for sense TGF-ß1; 146.88 ± 85.5 for antisense; 241.5 ± 181.5 for GL67-TGF-ß1 sense; and 88.4 ± 38.7 for saline. TGF-ß1 sense versus all other groups, p < 0.05, GL67-TGF-ß1 sense versus saline, p = 0.01. Rejection was significantly lower for TGF-ß1 sense versus saline, p = 0.04.
Conclusions. Endobronchial administration of naked plasmid achieves selective transfection of lung grafts. Using this strategy, TGF-ß1 reduces early lung allograft rejection.
Lung transplantation has proven to be a valid therapeutic option for a variety of end-stage pulmonary disease [1]. Lung allograft ischemia-reperfusion injuries as well as acute and chronic rejection continue to be challenges to the success of lung transplantation [1].
Gene therapy offers a potential strategy by genetic modulation of the biologic events that occur in ischemia-reperfusion injury and rejection [2, 4]. Administration of recombinant DNA to the lungs has been successfully performed by using viral and non-viral vectors delivered either via the vascular bed or the airway [3–7]. Both of these vector systems have shown limitations in their applicability being either immunogenic or pro-inflammatory [8–9]. Effective gene transfer to lung has also been demonstrated by administering the naked plasmid via the airway, thus suggesting that potentially toxic vectors may be unnecessary for gene transfer [6, 10, 11].
The aim of this study was to investigate the airway as a route for selective administration of recombinant genes to rat lung grafts. Naked plasmid or liposome-plasmid complex were used. The reporter gene for chloramphenicol acetyl transferase (CAT) was administered to isograft to determine transfection efficiency, and the murine transforming growth factor beta 1 (TGF-ß1) cDNA in allograft to achieve effective immunomodulation.
Material and methods
Animals
Inbred male F344 rats (Harlan Sprague Dawley Inc, Indianapolis, IN) and Brown Norway rats with a weight range of 250 to 300 gm were used. All animal procedures were approved by the Animal Studies Committee at Washington University. Animals received humane care in compliance with "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 National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 85-23, revised 1985).
Plasmids
The plasmid pCF1-CAT, kindly provided by Drs Ronald K. Schule and Nelson Yew (Genzyme Corporation, Framingham, MA) consists of the human cytomegalovirus (CMV) immediate-early promoter and enhancer followed by the tripartite leader from adenovirus, a hibrid intron, the CAT cDNA and a polyadenylation signal from bovine growth hormone.
The plasmid pMP6A-TGF-ß1 (sense and antisense) is an adeno-associated virus-based plasmid encoding the murine TGF-ß1 (sense and antisense) precursor gene under the control of the CMV early promoter, kindly provided by Dr Jonathan Bromberg (University of Michigan). The pMP6A consists of a cassette containing the terminal repeat elements of the adeno-associated virus 5' and 3' to the CMV immediate early promoter, a hybrid intron, and the SV40 polyadenylation signal sequence.
Liposomal vector
GL #67, an amphiphile consisting of a hydrophobic lipid anchor (cholesterol) linked to a spermine headgroup in a "T shape" configuration in a 1:2 ratio with the co-lipid, L-dioleoyl phosphatidyl-ethanolamine (DOPE) was kindly provided by Drs Nelsen Yew and Ronald Schule (Genzyme Corporation). Before use, the dried lipid films were rehydrated with saline solution, vortexed for 10 seconds, placed on ice for 10 minutes, and vortexed again for 2 minutes. Equal volumes of resuspended GL-67/DOPE and plasmid DNA were mixed and incubated at room temperature for 30 minutes. Final concentrations were 1 mmol/l cationic lipid and 4 mmol/L plasmid DNA.
Gene delivery
After anesthesia and oro-tracheal intubation with a 14-gauge catheter, a selective cannulation of the left main bronchus was performed over a guide wire with PE20 tubing. The position of the guide wire was confirmed by fluoroscopy or by direct assessment of the right hilum. Afterwards, 0.1 ml of saline suspension of plasmid alone or liposome-plasmid complex containing 130 µg of DNA was instilled. The animals were then placed in the left decubitus position and ventilated for 10 minutes. Thereafter they were either awakened or underwent left lung harvest according to the experimental design.
Experimental design
The study was divided into two parts based on the complementary DNA used: CAT or TGF-ß1.
Phase I: non-transplant setting
F344 rats underwent selective left lung gene transfection by either plasmid pCF1-CAT (n = 4) or by lipid-plasmid complex GL67:DOPE-pCF1-CAT (n = 4). A third group (n = 4) received saline solution and served as the control. The correct position of the guide wire and cannula within the left main bronchus was confirmed by fluoroscopy. Animals were sacrificed 2 days after treatment. CAT protein expression was assessed in the left lung, in the heart, and in the liver using a CAT enzymatic linked immunosorbent assay (ELISA). Histology was assessed by a blinded observer (pathologist—J.H.R.).
Phase I: isogenic transplant setting
F344 rats underwent isogenic orthotopic left lung transplant [3]. The donor left lung was transfected according to the protocol with either the plasmid pCF1-CAT alone (n = 5) or with the lipid-plasmid complex GL67:DOPE:pCF1-CAT (n = 5). The correct position of the cannula within the left main bronchus was determined by direct assessment of the right hilum. The recipients were sacrificed on the second postoperative day. ELISA was used to quantify CAT protein expression in the isograft right native lung, in the heart, and in the liver.
Phase II: allograft acute rejection
Brown Norway rats (RT1n) served as donors and F344 rats (RT1lvl) as recipients in four study groups. This strain combination was chosen because of the strong major and minor histocompatibility locus mismatch resulting in a well-documented complete lung graft rejection known to occur within the 5th postoperative day in control recipients without immunosuppression. The donors underwent selective left main bronchus gene delivery after laparotomy and sternotomy were performed. Gene delivery was performed by instillation of: pMP6A-TGF-ß1 sense (n = 6); pMP6A-TGF-ß1 antisense (n = 5); lipid-plasmid complex GL67:DOPE; and pMP6A-TGF-ß1 (n = 10). A fourth group (n = 10) received normal saline solution and served as a control. Correct cannulation of the left main bronchus was confirmed by intraoperative direct assessment.
In all groups, no immunosuppressive drugs were used and recipients were sacrificed on the 5th postoperative day according to previous studies [3, 4]. Arterial oxygenation was assessed after 5 minutes of left lung mono-ventilation (100 strokes/min; FiO2 100%; tidal volume 1.5 ml). A blinded observer (pathologist—J.H.R.) scored rejection according to the 1995 revision of the working formulation for the classification of pulmonary allograft rejection [12]. Vascular and airway rejection scores ranged from 0 (no rejection) to 4 (complete destruction of the allograft). Reverse transcriptase polymerase chain reaction (RT-PCR) was used to confirm transfection of the murine TGF-ß1 within rat lung parenchyma. ELISA was used to quantify TGF-ß1 protein expression.
Assessments
RT-PCR
Total RNA was extracted from the allograft lung by using the RNeasy Midi Kit (Quiagen, Santa Clarita, CA). One microgram of total RNA was reverse transcribed with random hexamers and Moloney leukemia virus reverse transcriptase in a total volume of 40 µl using Gene Amp RNA PCR Core Kit (Perkin-Elmer, Roche Molecular Systems, Inc, Branchburg, NJ) according to the manufacturer’s directions. The reaction mixture was then divided into two parts and amplified with specific rat ß-actin and murine TGF-B1 primers. After a 2 minute denaturation at 95°C, samples underwent PCR (denaturation at 95°C for 15 seconds, primer annealing at 50°C for 15 seconds, and extension for 15 seconds at 72°C). To stop the reaction within the linear range, PCR amplification of ß-actin was stopped after 25 cycles and TGF-ß after 40 cycles. The PCR reaction was analyzed by electrophoresis of 10 µl of the reaction mixture through a 2.5% agarose TBE (Tris/boric acid/EDTA) gel.
Primer sequences were: 5'-CTACAATGAGCTGCGTGTGG-3' for ß-actin forward and 5'-ATGGCTACGTACATGGCTGG-3' for ß-actin reverse [13]; 5'-TTGCAGAGATTAAAA-3' for murine TGF-ß1 forward, and 5'-TCCACGTGGAGTTTG-3' for murine TGF-ß1 reverse. Oligonucleotide synthesis was done by the Nucleic Acid Chemistry Laboratory (Washington University, St. Louis, MO). The length of the amplified cDNA regions was 138 bp for ß-actin and 236 bp for the murine TGF-ß1.
ELISA for CAT and TGF-ß1
CAT protein was extracted by homogenizing lung in Tris-EDTA. Homogenates underwent three cycles of freeze thaw: 5 minutes at -80°C in a dry ice methanol bath and 5 minutes in 37°C water bath. The homogenate was then centrifuged at 10,000 rpm for 15 minutes and the supernatant was used to perform CAT ELISA (CAT ELISA kit by Boehringer-Mannheim, Mannheim, Germany). Total protein concentration of the supernatant was calculated by using the BCA Protein Assay kit (Pierce, Rockford, IL).
TGF-ß1 protein was extracted from lung tissue as previously described [4]. Briefly, the lower third of the allograft lung was placed in 4 ml of cold acid-ethanol solution containing protease inhibitors (Complete Mini tabs, Boehringer-Mannheim). Five micrograms per milliliter of pepstatin was added and was immediately homogenized for 1 to 2 minutes at 4°C with a Tissue Tearer. After overnight extraction at 4°C, the extracts were clarified by centrifugation at 10,000g for 10 minutes and the resulting supernatants were dialyzed against 4 mM HCl at 4°C using a 6,000 to 8,000 MW-cutoff Spectrapore dialysis tubing. The ELISA kit used in this study (Quantikine human TGF-ß1, DB100, by R&D Systems, Minneapolis, MN) is crossreactive for human rat and mouse TGF-ß1.
Statistical analysis
Parametric data was analyzed after logarithmic correction by one-way analysis of variance (ANOVA), multiple comparisons were made with Fisher’s test. Non-parametric data was analyzed using Kruskal-Wallis rank test and then the Mann-Whitney test along with the Bonferroni correction. Differences were considered significant when the p value was less than 0.05.
Results
Phase I
CAT protein expression quantified by ELISA within the lungs both in the non-transplant and transplant is shown in Figure 1. In the non-transplant, setting a greater protein expression was noted with naked plasmid compared liposome-plasmid complex, while in the transplant setting, the CAT protein expression was comparable for either vector system. The CAT protein expression obtained by the naked plasmid was significantly greater in the non-transplant setting compared to the transplant setting.
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No difference in the histological inflammatory response was noted between the two groups.
Phase II
The recipients were sacrificed on the 5th postoperative day when, according to previous studies, acute rejection is evident.
Arterial oxygenation tension after 5 minutes of mono-ventilation of the allograft is shown in Figure 2. Improved gas exchange was observed in the lungs that were transfected with the sense TGF-ß1 constructs. Superior gas exchange was noted in the group that received the naked plasmid.
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Comment
The fascinating potential of gene transfer to lung grafts is currently being investigated as a means to affect ischemia reperfusion injury and acute rejection [2–4]. Promising results have been obtained although optimal conditions of safe, efficient, and effective transfection of pulmonary parenchyma have not yet been established.
In this study, we investigated within the lung transplant setting the use of non-viral vector systems, liposome-plasmid complex, or naked plasmids, delivered by endobronchial catheter instillation.
In the first part of the study, we assessed the transfection efficiency by administering the CAT reporter gene in both the non-transplant and transplant setting. Naked CAT plasmid or liposome-plasmid complex resulted in comparable CAT protein production in the transplant setting, while in the non-transplant setting, protein production was greater with naked plasmid alone. We chose to rehydrate the liposomes with saline solution and not with water as suggested by the manufacturer. This was done to avoid the administration of water to the lungs. A comparison experiment between the two-rehydration methods was conducted and no difference in the transfection efficiency was noted in our setting (data not shown).
Similar and contrasting findings observed in the present study have been described in the literature. Tsan and colleagues and Meyer and coworkers [10, 11] have shown that the transgene expression of reporter genes obtained after intratracheal instillation of naked plasmid or liposome-plasmid complex was comparable. Zabner and colleagues [6] also demonstrated that airway transfection using naked plasmid or liposome-plasmid complex encoding CFTR had comparable beneficial effects in cystic fibrosis. Zabner and coworkers’ study employed similar plasmids and liposomes as used in our study. Griesenbach and associates and Yoshimura and coworkers [14, 15] described contrasting results, demonstrating, respectively, a 40-fold and a five-fold increment of efficiency using liposome vector compared to naked plasmids. The different conditions and materials used in these reports may explain the different results. In our setting, the liposome GL67 may have a low efficiency. Furthermore, liposomes may be inhibited by surfactant as observed in vitro [16, 17], or endobronchial catheter instillation may favor naked plasmid delivery [11]. The lower protein production observed using naked DNA within the transplant setting compared to the non-transplant setting may be secondary to degradation of the naked plasmid during the preservation period [11].
No CAT protein was observed in hearts and livers both in the non-transplant and transplant setting, and in the native right lungs of the recipients, therefore indicating that organ selectivity is obtained by using the airway route of administration.
Based on these findings, we investigated the potential immunomodulating effect of naked TGF-ß1 cDNA administered via the airway in a lung allograft acute rejection model [3]. TGF-ß1 is a multifunctional molecule with a very potent immunomodulator function expressed by suppressing the proliferation of B and T cells, antagonizing inflammatory cytokines such as IL-1ß, TNF
, or IFN
, and inhibiting natural killer cells [18–20].
Previous experimental studies in the heart or lung allograft acute rejection setting have shown beneficial effects of TGF-ß1 gene transfer [2–4]. In particular, ex vivo lung allograft retrograde vascular injection or pulmonary artery transfection of TGF-ß1 gene transfer by liposome vectors has shown beneficial effects [3, 4]. In the second part of this study, murine TGF-ß1 cDNA was administered via the airway to lung allografts maintaining the same experimental conditions described in the first phase.
Attenuation of lung allograft acute rejection was noted following administration of naked DNA or liposome-plasmid complex for TGF-ß1. The effective role of the TGF-ß1 sense plasmid was confirmed by the negative results obtained using the antisense plasmid and by the RT-PCR that documented the transgene expression of the transfected murine TGF-ß1 cDNA. Naked TGF-ß1 sense plasmid provided superior outcome as assessed by arterial oxygenation and rejection score thus suggesting better efficiency than the liposome plasmid construct.
It is difficult to explain the apparent difference in magnitude in amelioration of the functional outcome compared to the rejection score. It should be taken into consideration that histologic examination is a crude method to analyze the rejection process.
The TGF-ß1 protein levels within the groups that received sense TGF-ß1 cDNA proved to be significantly lower compared to the control group, a result that may support the suggested role of TGF-ß1 as a marker of rejection [21].
The results obtained from the TGF-ß1 ELISA do not reflect transgene expression of transfected murine derived TGF-ß1 cDNA but overall TGF-ß1 protein levels: endogenous and transgene protein. This was an obstacle to documentation of specific transgene expression levels of murine cDNA. Therefore it was not possible to evaluate the performance of the adeno-associated virus-based plasmid which served as vector for the murine TGF-ß1 cDNA in the second phase of the study. A significant better performance of the adeno-associated virus-based plasmids, both for levels and duration of transgene expression over a similar plasmid lacking the inverted terminal repeat of the adeno-associated virus at each end of the expression cassette, has been demonstrated in vitro [22].
It is our contention that an increment of the TGF-ß1 protein expression in the first postoperative days prior to the onset of the acute rejection process is the key to the beneficial effect observed in this study.
In conclusion, endobronchial instillation of naked plasmid to lung grafts may provide an organ selective and effective transfection system to lung allografts. In this setting, naked plasmid may be a better option than liposomes as vectors for gene transfer. No toxicity has been related to naked plasmid administration, therefore the relative limitation of transient transgene expression may be overcome by nontoxic repeat administrations [10, 11].
Acknowledgments
The authors thank Roberto Chiesa, PhD, for sharing his knowledge in genetics, Richard B. Schuessler, PhD, for the statistical consultation, Kathleen Grapperhaus for the technical assistance; and Dawn Schuessler for secretarial support.
This work is supported by the NIH grants RO1 HL-41281 and RO1 HL56643.
References
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