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Eur J Cardiothorac Surg 2006;29:772-778
© 2006 Elsevier Science NL

Comparison of two gene transfer models for the attenuation of myocardial ischemia–reperfusion injury following preservation for cardiac transplantation

Haitham Abunasra * , Ryszard T. Smolenski, John Yap, Jay Jayakumar, Mary Sheppard, Magdi H. Yacoub

Heart Science Centre, Imperial College at Harefield Hospital, Harefield, Middlesex UB9 6JH, UK

Received 12 September 2005; received in revised form 11 December 2005; accepted 13 December 2005.

* Corresponding author. Address: 67 Glenfrith Close, Leicester LE3 9QQ, UK. Tel.: +44 1162877711. (Email: haitham7{at}hotmail.com).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 Appendix A
 References
 
Objective: Two models of ex vivo gene transfer were compared by examining the protective effect of adenovirus-mediated transfection of a free radical scavenger superoxide dismutase (SOD) during experimental ischemia–reperfusion mimicking preservation for cardiac transplantation. Methods: Donor rat hearts (n = 6 per group) were infused (subgroups IA and IB) or continually perfused (subgroups IIA and IIB) with solution containing adenoviral vector carrying ß-galactosidase (subgroups IA and IIA) or Mn-SOD (subgroups IB and IIB) over 5 s with 1 h storage and 15 min, at 4 °C, respectively. Hearts were then implanted heterotopically into the abdomen of recipient rats. Four days later, transplanted hearts were collected, connected to Langendorff perfusion apparatus and subjected to 6 h of ischemia followed by 1 h of reperfusion. Cardiac function was evaluated using intraventricular balloon at the beginning of Langendorff perfusion and following ischemia–reperfusion. Results: Blue staining from hydrolyses of X-gal by ß-galactosidase was confirmed in AdLacZ transduced hearts. Immunoreactivity with anti-human Mn-SOD antibody then staining was positive in AdMnSOD-transduced hearts. Percent recovery of preischemic left ventricular developed pressure (LVDP) increased from 55.9 ± 3.1% to 67.3 ± 6.2% (P = 0.048) and from 58.0 ± 8.0% to 78.9 ± 6.0% (P < 0.001) in subgroups IA, IB, IIA and IIB, respectively. The difference in LVDP recovery between treatment groups of the two transfection methods (IB vs IIB) was significant (P = 0.044). Conclusion: Adenoviral Mn-SOD ex vivo delivery using continuous myocardial perfusion is superior to bolus infusion in the attenuation of myocardial ischemia–reperfusion injury.

Key Words: Adenoviral gene transfer • Transplantation • Ischemia–reperfusion • Superoxide dismutase


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 Appendix A
 References
 
Increased formation of reactive oxygen species following oxidative stress plays a major part in the pathogenesis of myocardial ischemia–reperfusion injury [1,2]. Superoxide dismutase (SOD) catalyses the dismutation of superoxide anion to hydrogen peroxide that is further processed by other antioxidant enzymes leading to neutralisation of radical species [3,4]. Three isoforms of SOD exist; copper/zinc SOD (Cu/Zn-SOD) which has a cytoplasmic location, extracellular SOD (EC-SOD) present outside the cell and manganese SOD (Mn-SOD) which is found in the mitochondrial matrix. Modulation of the last isoform is a critical determinant in the tolerance of the heart to oxidative stress [5]. We demonstrated previously that ex vivo gene transfer of Mn-SOD can attenuate experimental myocardial ischemia–reperfusion injury [6].

Continuous hypothermic perfusion of donor hearts compared with hypothermic immersion storage can be used for preservation of donor organs [7,8]. Therefore, a modified hypothermic perfusion technique could be applied for gene delivery to donor hearts. It was shown that when adenovirus is used as a vector, this technique would result in more efficient transgene expression compared with that induced by a single bolus injection [9]. Pellegrini and colleagues [9] showed that adenoviral hypothermic perfusion model provided an 11–14-fold increase in transgene (control gene) expression compared with the high-pressure bolus injection. Use of a free radical scavenger in a similar protocol has not been examined before. This study compares the two methods of adenoviral Mn-SOD gene delivery in the rat heart on myocardial functional recovery following global ischemia–reperfusion injury, mimicking preservation for cardiac transplantation, to identify the optimal method for use in this setting.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusion
 Appendix A
 References
 
2.1 Animals
Male Sprague–Dawley rats (weight 250–300 g) were used as donors and recipients in this study. All animals received humane care in compliance with the European Convention on Animal Care. This study was approved by the Institutional Ethics Committee on Animal Research.

2.2 Adenoviral vector
A serotype 5 adenovirus encoding for nonnuclear targeted Escherichia coli ß-galactosidase under the control of the cytomegalovirus promoter was used in the control group (AdCMVLacZ, provided by James Wilson, Institute for Gene Therapy, University of Pennsylvania, PA, USA). This vector has been rendered replication defective by replacing the entire E1a and most of the E1b regions of the adenoviral genome with the complementary DNA expression cassette. Mn-SOD recombinant adenoviral construct was generated using a previously described method. Briefly, Mn-SOD constructs were generated by cloning of an EcoRI/PvuII fragment from the pRK5 Mn-SOD construct [10]. Recombinant adenoviral plasmid construct were generated by cloning transgene into pAd.CMVlink, which contains the CMV enhancer/promoter and an SV40 polyadenylation site for efficient expression of the transgene [11]. Recombinant virus was generated by cotransfection of NheI-cut pAd plasmid with ClaI-cut Ad5.sub360 (E3-deleted) viral DNA [12]. After transfection, plates were overlaid with agar, and initial plaques were harvested for screening by enzymatic activity. This recombinant virus was screened for Mn-SOD activity by secondary infection on 293 cells. Initial plaques that expressed functional enzyme were further purified through two subsequent rounds of plaque purification. Viral titre was determined by assessing PFU on 293 cells.

2.3 Experimental groups
Rats were divided into two groups (I and II, n = 12 each) according to the method of gene delivery used. Each group had two equal subgroups: one had the LacZ gene transfected as control (A) and in the other, treatment gene Mn-SOD was used (B). Hearts collected from donor rats were globally transfected using bolus infusion (Group I) or continuous perfusion (Group II) method and transplanted into the abdomen of recipient rats as described in detail below. Four days later, necessary for gene expression, transplanted hearts were excised connected to the Langendorff system and subjected to cold cardioplegic arrest and reperfusion accompanied by myocardial function monitoring. Analysis of samples collected at the end was done using immunostaining (Fig. 1 ).


Figure 1
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  		Fig. 1. Experimental protocol. Four days after gene transfection, hearts were subjected to 6 h of global ischemia and 1 h of reperfusion. Hearts were assessed for function using intraventricular balloon and then subjected to immunohistochemical analysis.

 
2.4 Donor operation and gene transfer
Donor rats (250–275 g) were anaesthetised with sodium pentobarbital (50 mg/kg, intraperitoneally). A median laparotomy was performed to expose the abdominal aorta. After injection of 200 U of aqueous heparin into the inferior vena cava (IVC), the aorta was cannulated with a 24-gauge cannula, and the heart was arrested with an infusion of 30 mL cold University of Wisconsin Solution (UWS), 4 °C, into the aorta over 5 s. A median sternotomy was performed, venae cavae and pulmonary veins were ligated with 4-0 silk and the heart harvested and stored in the same cardioplegic solution at 4 °C.

In Group I: 350 µL of UWS containing viral titre, 1.0 x 109 PFU/mL of AdCMVLacZ (IA) or AdCMVMnSOD (IB), was infused as a high-pressure bolus over 5 s into the coronary artery through the aortic root. The pulmonary artery was clamped during the infusion, and the virus was not flushed out at the end of 60 min of cold storage before performing the surgical procedure. In Group II, 5 mL of UWS containing viral titre, 1.0 x 109 PFU/mL of AdCMVLacZ (IIA) or AdCMVMnSOD (IIB), was circulated through the coronary vasculature of the donor heart for 15 min by means of a peristaltic pump (Rainin, Emeryville, CA, USA). The viral solution was perfused into the donor organ through the cannula inserted into the aorta and was collected by a 14-gauge catheter placed into the pulmonary artery. Both catheters were connected by means of polyvinyl chloride tubing to the vial containing the viral solution. The flow rate was 0.75 mL/min. During the perfusion period, the container with the heart and the vial with the vector were kept on ice, and temperatures of both solutions did not exceed 4 °C.

2.5 Heterotopic heart transplantation
Heterotopic abdominal heart transplantation was performed using standard microsurgical techniques [13]. Rats (275–300 g) were anaesthetised by administration of intraperitoneal pentobarbital (70 mg/kg). The donor hearts were transplanted into the recipients by end-to-side anastomoses of the aorta and the pulmonary artery to the abdominal aorta and inferior vena cava, respectively, using 8-0 monofilament sutures. During surgery, the heart was wrapped in gauze and kept cold using topical ice-cold saline solution. Mean duration of all transplant procedures was 37.4 ± 5.6 min. Postoperatively, all rats recovered with oxygen in a warm environment. Viability of the grafts was verified daily by palpation of the beating transplanted heart.

2.6 Global ischemia–reperfusion
On the fourth day after gene transfer, animals were anaesthetised with diethyl ether and anticoagulated by intravenous injection of heparin (500 U). Transplanted hearts were quickly excised and perfused with modified Krebs–Henseleit buffer (120.0 mM NaCl, 4.5 mM KCl, 20.0 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgCl2, 2.5 mM CaCl2 and 10.0 mM glucose; gassed with 95% O2 + 5% CO2 to obtain pH 7.4 at 37 °C) at a pressure equal to 1 mH2O by means of a Langendorff apparatus. A thin-wall balloon was inserted into the left ventricle through the left atrium to monitor left ventricular pressure and control left ventricular volume. After stabilization, left ventricular developed pressure (LVDP), maximum dP/dt (max dP/dt), minimum dP/dt (min dP/dt) and rate–pressure product (RPP) were measured with LV diastolic pressure stabilized at 10 mmHg (RPP expresses cardiac work over specific time). Hearts were then subjected to global ischemia by infusion of cold (4 °C) crystalloid (St. Thomas’ No. 1) cardioplegia for 6 h followed by 1 h of reperfusion. Then, the same measurements of the LV were repeated with the balloon inflated to preischemic volume.

2.7 X-gal staining
Hearts from subgroups IA and IIA were dissected, embedded in OCT medium (Miles, Elkhart, IN, USA) and frozen in liquid nitrogen. Frozen sections (6 µm thick) were fixed in 2% paraformaldehyde, 0.125% glutaraldehyde in PBS for 5 min, washed three times in PBS with 2 mM magnesium chloride then incubated in three changes of PBS containing 2 mM magnesium chloride, 0.01% sodium deoxycholate and 0.02% NP-40. Sections were then incubated in staining buffer (30 mM potassium ferrocyanide and 30 mM potassium ferricyanide in PBS containing 2 mM magnesium chloride, 0.01% sodium deoxycholate and 0.02% NP-40) for 2 min prior to incubation in fresh staining buffer containing 1 mg/mL 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) and incubated in a moist chamber overnight at 37 °C. Subsequently, sections were rinsed in PBS and counterstained with neutral red and rinsed in water before mounting. Blue-stained cells indicated the presence of ß-galactosidase expression.

2.8 Immunohistochemical staining
Midventricular cross-sections of the transplanted hearts from subgroups IB and IIB were embedded in OCT medium (Miles) and frozen in liquid nitrogen. Frozen sections were cut at 25 (m intervals, fixed for 10 min in cold acetone (4 °C), fan-dried for 10 min and further fixed in 1% paraformaldehyde/EDTA for 3 min. Endogenous peroxidase activity was blocked with 0.1% sodium azide/0.3% H2O2 for 10 min. Incubating sections with 5% goat serum/PBS–Tween 20 blocked non-specific protein binding sites. Samples from subgroups IB and IIB then had 1:200 of anti-Mn-SOD monoclonal antibody (K90096C) (BioDesign, UK) added and were incubated for 60 min at room temperature. After rinsing, biotinylated rabbit anti-mouse F(ab')2 1:300 was added for 20 min. After further incubation for 20 min with 1:1000 sheep/goat peroxidase (M15345), the slides were incubated for 30 s in 0.1 M sodium acetate buffer, pH 5.2. Then, were placed in 3-amino-9-ethylcarbazole substrate solution and incubated for 15 min at room temperature, counterstained in mercury-free haematoxylin for 1 min and further rinsed for 3 min in cold running tap water before being mounted.

2.9 Statistics
Values are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used followed by Bonferroni test to indicate individual significant differences. A value of P < 0.05 was considered as a significant difference.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusion
 Appendix A
 References
 
There was no technical failure or operative death in the 24 consecutive gene transfection experiments in the study. Positive cardiomyocyte cytoplasm staining with X-gal can be seen in cardiomyocytes from the LacZ transfected subgroups (IA and IIA), but has not been observed in non-transfected animals (Fig. 2 ). Immunohistochemical staining for Mn-SOD showed endothelial and cardiomyocyte expression of Mn-SOD in subgroups IB and IIB hearts (Fig. 3 ). A small increase in the number of inflammatory cells in IIB compared to IB hearts was observed but not measured (immunological reaction to the adenoviral vector).


Figure 2
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  		Fig. 2. ß-gal transgene expression. Demonstration of ß-galactosidase transgene expression by histochemical staining with X-gal in heart transfected with AdLacZ in subgroups IA and IIA. Myocytes appear positive for ß-gal. Original magnification 600x.

 

Figure 3
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  		Fig. 3. Mn-SOD immunohistochemical staining. Immunohistochemical staining of a midventricular section of heart transfected with AdMnSOD in subgroups IB and IIB, using monoclonal antibody to Mn-SOD (counterstained with mercury-free haematoxylin). Expression is present in cardiomyocytes. Original magnification 400x.

 
In Langendorff perfusion experiments, no significant difference was seen before ischemia among the groups (n = 6 in each group, Table 1 ) in terms of LVDP, max dP/dt, min dP/dt and RPP. Percent recovery of LVDP after global ischemia (4 °C, 6 h) is shown in Fig. 4 . The percent recovery of LVDP after ischemia in the control transfected hearts reached the peak value 55.9 ± 3.1% in IA and 58.0 ± 8.0% in IIA hearts after reperfusion. In comparison, a statistically significant improvement of the percent recovery of LVDP was observed in the subgroups transfected with Mn-SOD, which reached a peak level 67.3 ± 6.2% in IB (P = 0.048 vs IA) and 78.9 ± 6.0% in IIB (P < 0.001 vs IIA) after reperfusion. The difference between subgroups IIB and IB was statistically significant (P = 0.044).


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  		Table 1. Cardiac function parameters before ischemia during Langendorff perfusion
 

Figure 4
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  		Fig. 4. Recovery of LVDP after ischemia. The isolated hearts in four experimental groups were subjected to 6 h of cold (4 °C) global ischemia followed by 1 h reperfusion. Significantly better recovery of LVDP after ischemia was shown in subgroups IB and IIB when compared to subgroups IA and IIA, respectively. Subgroup IIB showed best significant recovery. * P = 0.048 versus IA, P < 0.001 versus IIA and @ P = 0.044 versus IB, n = 6 in each group. Data are expressed as percentage of LVDP before ischemia. All values are expressed as mean ± SD.

 
Percent recoveries of max dP/dt and min dP/dt after global ischemia are shown in Fig. 5 . Percent recovery of max dP/dt after reperfusion in IIB and IB hearts showed a significant difference (P = 0.049). Both IIB and IB hearts showed important recovery in max dP/dt when compared to control hearts (IIB vs IIA, P < 0.001 and IB vs IA, P = 0.018). IIB hearts showed a significantly improved percent recovery of min dP/dt following ischemia–reperfusion (P = 0.019 vs IIA). However, the percent recovery in subgroup IB was not significant (P = 0.079 vs IA). The difference in min dP/dt recovery following reperfusion showed no statistical significance between subgroups IIB and IB (P = 0.960). Values for min dP/dt were 51.1 ± 2.9% (IA), 66.3 ± 8.7% (IB), 55.7 ± 13.1% (IIA) and 74.5 ± 7.5% (IIB). Max dP/dt values were 49.7 ± 7.2%, 64.2 ± 7.8%, 52.2 ± 5.0% and 76.9 ± 6.8% in subgroups IA, IB, IIA and IIB, respectively.


Figure 5
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  		Fig. 5. Recovery of max dP/dt and min dP/dt after ischemia. Recovery of max dP/dt and min dP/dt after 6 h of cold global ischemia and 1 h reperfusion. Data are expressed as percentage of basal max dP/dt and min dP/dt before ischemia. * P = 0.018 versus IA, P < 0.001 versus IIA, @ P = 0.049 versus IB, ** P = 0.019 versus IIA, n = 6 in each group. All values are expressed as mean ± SD.

 
In Fig. 6 , subgroup IIB hearts showed significantly better percent recovery of preischemic RPP compared with subgroup IIA (P < 0.001) and subgroup IB hearts (P = 0.01). The difference between subgroups IB and IA was not significant (P = 0.108). The values for RPP percent recoveries were IA = 46.8 ± 4.3%, IB = 56.5 ± 6.7%, IIA = 48.7 ± 5.5% and 70.3 ± 6.8% for subgroup IIB hearts. Although we did not use pacing on the Langendorff system, heart rate was relatively consistent in the preischemic period in all four subgroups studied here.


Figure 6
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  		Fig. 6. Recovery of RPP after ischemia. Recovery of RPP after 6 h of cold global ischemia and 1 h reperfusion. Data are expressed as percentage of RPP measured before ischemia. P < 0.001 versus IIA and @ P = 0.01 versus IB, n = 6 in each group. All values are expressed as mean ± SD.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusion
 Appendix A
 References
 
This study demonstrated that adenoviral gene transfer of Mn-SOD using continuous myocardial perfusion method as compared to bolus injection for gene delivery results in further improvement in postischemic recovery of mechanical function in the donor hearts following a protocol mimicking preservation of hearts for transplantation.

The important role of Mn-SOD in protecting hearts against detrimental effects of ischemia–reperfusion injury has been clearly shown by work with transgenic mice overexpressing Mn-SOD [14]. These mice had better postischemic recovery of ventricular function after a period of ischemia and reperfusion. However, this protocol used a short period of ischemia, whereas this study used a prolonged period of hypothermic ischemia after cardioplegic arrest mimicking clinical donor heart preservation. Furthermore, in a transgenic animal, the heart is genetically altered to overexpress Mn-SOD, so that it may adapt itself and develop a phenotype different from that of the natural heart. Therefore, the present model with gene transfection might be more suitable for investigating the effectiveness of Mn-SOD in the setting of cardiac transplantation.

There are three isoforms of SOD: Cu/Zn-SOD which has a cytoplasmic location, EC-SOD found in extracellular compartment and Mn-SOD which is found in the mitochondrial matrix [4]. Mn-SOD dismutases superoxide to form hydrogen peroxide, which in turn inactivated by glutathione catalysed by glutathione peroxidase or catalase [3,4]. The resulting mitochondrial damage from oxidant excessive production occurs once antioxidant enzyme systems are overwhelmed. Asimakis and associates [5] have shown that myocardial postischemic functional recovery is more sensitive to partial deficiency of Mn-SOD than that of Cu/Zn-SOD in transgenic mice, which stresses the importance of mitochondrial SOD in this setting and is consistent with observed significant recovery of myocardial function following ischemia–reperfusion injury, in the current study.

Woo and associates [15] found that adenoviral gene transfer, using intrapericardial delivery method, of SOD and catalase attenuates postischemic contractile dysfunction. Intracoronary route, used in our study, would result in wider distribution of gene transfection in all layers of the donor heart. Li and associates [16] showed that in vivo adenoviral gene transfer of membrane-bound EC-SOD through the intravenous route provides the heart with substantial protection against myocardial infarction. However, this method of gene delivery would result in systemic distribution of adenovirus with possible toxic effects. Direct intramyocardial injection would result in uneven, localized, transgene expression with marked inflammatory response and is unsuitable in global gene transfection setting [17].

Continuous hypothermic perfusion of donor hearts compared with hypothermic immersion storage can be used for preservation of donor organs [7,8]. Therefore, a modified hypothermic perfusion technique could be applied for gene delivery to donor hearts. It was shown that when adenovirus is used as a vector, this technique would result in more efficient transgene expression compared with that induced by a single bolus injection [9]. Pellegrini and colleagues [9] showed that adenoviral LacZ hypothermic perfusion model provided an 11–14-fold increase in transgene expression compared with the high-pressure bolus injection. It allowed a 30-fold reduction in the viral dose compared with that found in other reports, without affecting the level of transgene expression. Brauner and colleagues [18] increased adenoviral vector uptake into the donor organ to 80% with the slow infusion technique, compared with 10% with bolus injection, when they studied the effect of gene transfer of immunosuppressive cytokines on cardiac allograft survival without myocardial functional measurements, Pellegrini and colleagues [9] achieved similar levels of gene transfection with lower dose of adenovirus with no significant inflammatory response. Similar protocol was adopted in the current study, however, the use of Mn-SOD gene transfer with subsequent pressure measurements has, to our knowledge, not been investigated before.

In this experiment, we compared functional myocardial recovery from global ischemia–reperfusion injury after Mn-SOD gene overexpression with either bolus infusion or continuous perfusion, as two different models of gene delivery. We found that the latter method resulted in significant recovery of LVDP, max dP/dt and RPP. This observation may be explained by superior Mn-SOD expression. Hypothermic continuous perfusion may have increased adenoviral exposure and then adhesion to cell membrane receptor [19], which augmented myocardial transgene expression of Mn-SOD and LacZ, proven here with increased immunohistochemical and X-gal staining, respectively, when compared with bolus infusion. This enhanced Mn-SOD protection, known to be in the mitochondria, may have played a significant role in the observed more significant functional recovery with hearts in this group. The importance of Mn-SOD in alleviating oxidative stress with myocardial reperfusion after ischemia may have been proven with the observed significant recovery of LVDP and max dP/dt in hearts transfected with Mn-SOD in each group.

One limitation of this study is lack of quantitative assessment of the efficiency of Mn-SOD expression using the two methods. However, accurate determination of Mn-SOD activity is challenging and in the present setting would be further complicated by non-uniform distribution of the activity. It is possible that lower activity but more uniformly distributed may exert more significant protection. We believe that the assessment of this treatment by the function of the heart rather than by Mn-SOD activity is more important, although the activity would be very important additional information.

In this study, we developed a novel system to compare two different models of gene delivery of an enzyme with a cardioprotective role in reperfusion injury, by studying the functional recovery of rat hearts on Langendorff perfusion following ischemia–reperfusion, mimicking human heart transplantation, in each model of gene transfer.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
Appendix A
 References
 
This study demonstrated that gene delivery using hypothermic continuous perfusion model had a potential to introduce supraphysiological levels of protective enzymes such as Mn-SOD, which resulted in significantly enhanced myocardial tolerance to reperfusion injury beyond that of intrinsic factors when compared to single bolus infusion method. This potentially useful technique should be considered in future adenoviral gene transfer work in experimental heart transplantation. Clinical relevance of this model needs to be investigated further.


   Appendix A
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 Appendix A
References
 
Conference discussion

Dr U. Fischer (Cologne, Germany): Did you ever think about, or do you have any data on, administration of antioxidants into the perfusion buffer? I think you have to show that the gene therapy is superior to that. Because we found comparable results in 1-hour ischemia pig or dog model by simply adding N-acetylcysteine as high potent antioxidant. Could you comment on that, please.

Dr Abunasra : Previous work that we have done using nitric oxide donors in the perfusate showed important levels of this enzyme in myocardial cells. Gene therapy offered theoretical advantage. And we found that gene transfer of Mn-SOD offered the most significant recovery following myocardial ischemia–reperfusion. This was a paper that was published last year.

Gene therapy may be more clinically applicable and usable in a human transplant model.

Dr W. Klepetko (Vienna, Austria): Could you tell us how long the perfusion period was in the continuous perfusion.

Dr Abunasra : Perfusion for 15 min using the peristaltic pump.

Dr Klepetko : And the perfusion was, during that period of time, for how long, how many minutes was it perfused?

Dr Abunasra : 15 min.

Dr Klepetko : Do you think this is the optimal time, or do you think that there is potentially another even better time period to perfuse?

Dr Abunasra : Pellegrini and colleagues showed that this time of perfusion provided an 11–14-fold increase in transgene expression compared with the high-pressure bolus injection. It allowed a 30-fold reduction in the viral dose compared with that found in other reports, without affecting the level of transgene expression.


   Acknowledgments
 
We want to thank John Engelhardt, University of Iowa (USA), for his donation of the AdMnSOD virus. This study was supported by the British Heart Foundation and the Magdi Yacoub Institute.


   Footnotes
 
{star} Presented at the joint 19th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 13th Annual Meeting of the European Society of Thoracic Surgeons, Barcelona, Spain, September 25–28, 2005.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 Appendix A
 References
 

  1. Griendling KK, Alexander RW. Oxidative stress and cardiovascular disease. Circulation 1997;96:3264-3265.
  2. Kloner RA, Przyklenk K, Whittaker P. Deleterious effects of oxygen radicals in ischemia/reperfusion: resolved and unresolved issues. Circulation 1989;80:1115-1127.[Abstract/Free Full Text]
  3. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ. Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem 2003;278(38):36027-36031.[Abstract/Free Full Text]
  4. Raha S, McEachern GE, Myrint AT, Robinson BH. Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase. Free Radic Biol Med 2000;29(2):170-180.[CrossRef][Medline]
  5. Asimakis GK, Lick S, Patterson C. Postischemic recovery of contractile function is impaired in SOD2+/– but not SOD1+/– mouse hearts. Circulation Feb 2002;105(8):981-986.[Abstract/Free Full Text]
  6. Abunasra H, Smolenski R, Yap J, Sheppard M, O’Brien T, Yacoub M. Multigene adenoviral therapy for the attenuation of ischemia–reperfusion injury after preservation for cardiac transplantation. J Thorac Cardiovasc Surg 2003;125:999-1006.
  7. Ferrera R, Larese A, Marcsek P, Guidollet J, Verdys M, Dittmar A, Dureau G. Comparison of different techniques of hypothermic pig heart preservation. Ann Thorac Surg 1994;57:1233-1239.[Abstract]
  8. Okada K, Yamashita C, Okada M, Okada M. Successful 24-hour rabbit heart preservation by hypothermic continuous coronary microperfusion with oxygenated University of Wisconsin solution. Ann Thorac Surg 1995;60:1723-1728.[Abstract/Free Full Text]
  9. Pellegrini C, Jeppsson A, Taner C, O’Brien T, Miller V, Tazelaar H, McGregor C. Highly efficient ex vivo gene transfer to the transplanted heart by means of hypothermic perfusion with a low dose adenoviral vector. J Thorac Cardiovasc Surg 2000;119:493-500.[Abstract/Free Full Text]
  10. Greenberger JS, Epperly MW, Jahroudi N, Pogue-Geile KL, Berry L, Bray J, Goltry KL. Role of bone marrow stromal cells in irradiation leukemogenesis. Acta Haematol 1996;96:1-15.[Medline]
  11. Engelhardt JF, Yang Y, Stanford-Perricaudet LD, Allen ED, Kosarsky K, Perridaudet M, Yankaskas JR, Wilson JM. Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1 deleted adenoviruses. Nat Genet 1993;4:27-33.[CrossRef][Medline]
  12. Logan J, Shenk T. Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection. Proc Natl Acad Sci USA 1984;81:3655-3659.[Abstract/Free Full Text]
  13. Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg 1969;57:225-229.[Medline]
  14. Chen Z, Siu B, Ho YS, Vincent R, Chua CC, Hamdy RC, Chua BH. Overexpression of MnSOD protects against myocardial ischemia/reperfusion injury in transgenic mice. J Mol Cell Cardiol 1998;30:2281-2289.[CrossRef][Medline]
  15. Woo YJ, Zhang JC, Vijayasarathy C. Recombinant adenovirus-mediated cardiac gene transfer of superoxide dismutase and catalase attenuates postischemic contractile dysfunction. Circulation 1998;98:II55-II260.
  16. Li Q, Bolli R, Qiu Y, Tang XL, Guo Y, French BA. Gene therapy with extracellular superoxide dismutase protects conscious rabbits against myocardial infarction. Circulation 2001;103(14):1893-1898.[Abstract/Free Full Text]
  17. Wang J, Ma Y, Knechtle SJ. Adenovirus-mediated gene transfer into rat cardiac allografts: comparison of direct injection and perfusion. Transplantation 1996;61:1726-1729.[CrossRef][Medline]
  18. Brauner R, Nonoyama M, Laks H, Drinkwater Jr. DC, McCaffery S, Drake T, Berk AJ, Sen L, Wu L. Intracoronary adenovirus-mediated transfer of immunosuppressive cytokine genes prolongs allograft survival. J Thorac Cardiovasc Surg 1997;114(6):923-933.[Abstract/Free Full Text]
  19. Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones E, Krithivas A, Hong JS. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997;275:1320-1323.[Abstract/Free Full Text]




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