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Ann Thorac Surg 2001;71:624-629
© 2001 The Society of Thoracic Surgeons

Original article: cardiovascular

A novel strategy of decoy transfection against nuclear factor-{kappa}B in myocardial preservation

Taichi Sakaguchi, MDa, Yoshiki Sawa, MDa, Norihide Fukushima, MDa, Motonobu Nishimura, MDa, Hajime Ichikawa, MDa, Yasufumi Kaneda, MDb, Hikaru Matsuda, MDa

a Department of Surgery, Course of Interventional Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
b Division of Gene Therapy Science, Osaka University Graduate School of Medicine, Osaka, Japan

Accepted for publication May 10, 2000.

Address reprint requests to Dr Sawa, Department of Surgery, Course of Interventional Medicine (E1), Osaka University Graduate School of Medicine, Yamada-oka 2-2, Suita, Osaka 565-0871, Japan
e-mail: sawa{at}surg1.med.osaka-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Nuclear factor-{kappa}B (NF{kappa}B) is critical for the transcription of multiple genes involved in myocardial ischemia-reperfusion injury. Therefore, we hypothesized that blocking NF{kappa}B would attenuate ischemia-reperfusion injury after prolonged myocardial preservation, resulting in an improvement in cardiac function.

Methods. Double-stranded oligodeoxynucleotides with a specific affinity for NF{kappa}B (NF{kappa}B decoy group) or a scrambled decoy group were transfected into rat hearts using a hemagglutinating virus of Japan–liposome method. After 16 hours of preservation in Euro-Collins solution at 4°C, the cardiac grafts were heterotopically transplanted into recipient rats of the same strain.

Results. Fluorescein isothiocyanate staining showed introduction of double-stranded oligonucleotides into the nuclei of endothelial cells and cardiomyocytes. After 1 hour of reperfusion the NF{kappa}B decoy group showed significantly higher degrees of recovery of left ventricular function as well as significantly lower levels of serum creatine phosphokinase, myocardial water content, tissue IL-8, and neutrophil infiltration than did the scrambled decoy group (p < 0.05).

Conclusions. Gene transfection of the NF{kappa}B decoy attenuates ischemia-reperfusion injury after prolonged heart preservation. As a result, this method appears to be a novel strategy for enhanced myocardial preservation.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Cardiac transplantation is now recognized as an established therapy for irreversible and end-stage heart failure. However, preservation of hearts is a major limitation in clinical heart transplantation. The main cause of death early after transplantation seems to be ventricular dysfunction, rather than rejection or infection, resulting from inadequate preservation [1]. Several strategies have been developed to improve organ preservation and to attenuate reperfusion injury to the graft [2–6]. However, they still remain inadequate to attenuate contractile and coronary dysfunction.

In the process of ischemia-reperfusion injury in the myocardium, endothelial cell activation is an initial step and plays a key role in the induction of adhesion molecules and cytokines [6–8]. In endothelial cell activation, nuclear factor {kappa}-B (NF{kappa}B) is the main transcription factor involved in up-regulation of pro-inflammatory gene products [9, 10]. The expression of these genes plays an important role in the initiation of inflammatory response by targeting circulating leukocytes at the site of inflammation. Therefore, blocking NF{kappa}B in the endothelial cells seems to be a useful strategy for attenuating ischemia-reperfusion injury in myocardium.

Synthetic double-stranded oligodeoxynucleotides (ODNs), as "decoy" cis elements, block the binding of nuclear factors to the promoter regions of targeted genes, resulting in the suppression of gene transactivation in both in vitro and in vivo models [9, 10]. We have previously demonstrated that an in vivo transfection of an NF{kappa}B decoy ODN using hemagglutinating virus of Japan (HVJ)-liposome as a vector attenuates ischemia-reperfusion injury in rat hearts [9, 10].

In this study, we hypothesized that transfection of NF{kappa}B decoy into endothelial cells and myocytes during hypothermic storage would enhance myocardial preservation by attenuating leukocyte-mediated reperfusion injury. The aim of this study was to examine the efficacy of decoy transfection for enhanced myocardial preservation.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Animals
Adult male Sprague-Dawley rats were used in this study. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).

Synthesis of ODN
The sequence of the phosphorothioate ODNs used (NF{kappa}B decoy ODN and scrambled decoy ODN) has previously been described [9, 10]. NF{kappa}B decoy ODN has been shown to bind to the NF{kappa}B transcriptional factor [11]. For histologic assessment of the efficiency of the ODN delivery, NF{kappa}B decoy ODNs were labeled with FITC on the 3' end using fluorescein-ODN phosphoramidite. These ODNs were provided by Greiner Japan (Tokyo, Japan).

Ex vivo transfer of ODN using the HVJ-liposome method
The HVJ-liposome was prepared as previously described [12, 13]. Sprague-Dawley rats (250 g) were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). After anticoagulation with heparin (200 USP units, intravenously), the hearts were arrested with cold crystalloid cardioplegic solution infused through the aorta. The hearts were excised and infused with 0.7 ml of HVJ-liposome–ODN complex by way of the ascending aorta with the vena cavae, pulmonary artery, and veins ligated. After incubation on ice for 10 minutes, the hearts were stored in 4°C Euro-Collins solution for 16 hours with the pulmonary artery open [10, 14].

Heterotopic cardiac transplantation
Recipient rats (350 g) of the same strain were anesthetized with the same way as the donor rats. For the procedure, each rat was placed on a warm water mat to maintain a rectal temperature of approximately 37°C (which was continuously monitored) and was continuously provided with 0.5 L/min of 100% O2. The left femoral artery and vein were cannulated for simultaneous monitoring of blood pressure and heart rate as well as volume substitution. The abdomen was opened with a midline incision and the donor heart was transplanted after hypothermic storage by means of a modification of the technique of Ono and Lindsey [15]. After completion of the anastomoses the heart was reperfused with blood in situ for 1 hour [6, 16].

Histologic assessment
A histologic assessment of the efficiency of the FITC-labeled ODN delivery by HVJ-liposome method in comparison with a direct transfer was performed. Rat hearts were transfected with FITC-labeled ODN with HVJ-liposome (10 µmol/L, n = 6) or without HVJ-liposome (10 µmol/L, n = 6). The hearts in each group were taken after 16 hours of preservation (n = 3 in each group) or after preservation followed by 1 hour of reperfusion (n = 3 in each group). They were frozen at –80°C, cut into thin sections (5 µm), and then examined by fluorescence microscopy.

Cardiac graft function and serum creatine phosphokinase
The performance of the transplanted heart was measured in situ after 1 hour of reperfusion. A thin-walled, latex balloon-tipped catheter was introduced into the left ventricle through the left atrial appendage to monitor left ventricular pressure. After 1 hour of reperfusion, heart rate (HR), left ventricular developed pressure (LVDP), and the maximal derivatives of left ventricular pressure (max dP/dt) were measured with the left ventricular end-diastolic pressure stabilized at 10 mm Hg. Crystalloid volume substitution (Ringer’s solution) was adjusted through the left femoral vein to maintain the mean arterial pressure of the recipient rat between 65 and 80 mm Hg for the reperfusion period. After completion of reperfusion, the hearts was excised and immediately frozen at –80°C for histologic assessment. Aortic blood samples were collected for the assay of serum creatine phosphokinase (CK).

Myocardial water content
To evaluate myocardial water content after the preservation followed by reperfusion, the basal region of the heart was taken and weighed. Next it was desiccated at 96°C for 24 hours and then reweighed. The myocardial water content was calculated by the following formula: Myocardial water content = (1 – dry weight/wet weight) x 100 (%).

Neutrophil adherence and tissue IL-8
Neutrophils in the myocardium were selectively stained using a naphtol AS-D chloroacetate esterase kit (Sigma Chemical, St. Louis, MO). The number of neutrophils was counted under light microscopy (magnification x100) in a blind manner. The total number of neutrophils in each section was summed and expressed as cell number per section. At least ten individual sections were evaluated per heart.

For measurement of tissue IL-8 production, the transfected hearts were excised after preservation followed by reperfusion and immediately frozen in liquid nitrogen. The samples were homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) and centrifuged. The concentration of tissue IL-8 was measured using ELISA kits (Immuno-Biological Laboratories, Gumma, Japan) according to the manufacturer’s recommendations.

Study group
Hearts were divided in two groups (n = 6 in each group): the NF group, transfected with the cis element "decoy" against the NF{kappa}B binding site; and the SD group, transfected with the scrambled decoy ODN.

Statistical analysis
All data are expressed as mean ± standard error of the mean (SEM). Scores were compared using an unpaired Student’s t test. A p value of less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Ex vivo transfection of NF{kappa}B decoy ODN
Fluorescence was detected in microvascular endothelial cells of hearts transfected with FITC-labeled ODN by the HVJ-liposome method after 16 hours of hypothermic preservation. Furthermore, after 1 hour of reperfusion, fluorescence was detected not only in vessels but also in myocytes. The fluorescence was localized mainly in the nuclei of cardiac myocytes and endothelial cells. On the other hand, there was very little fluorescence detected in those hearts transfected with FITC-labeled ODN by direct transfer after both preservation and reperfusion (Fig 1). These results are consistent with those of our previous study [9, 10].



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  		Fig 1. Ex vivo distribution of FITC-labeled oligonucleotides delivered by means of infusion into a coronary artery by the hemagglutinating virus of Japan–liposome method (A, B) or direct transfer of FITC-labeled oligonucleotides (C, D). With the aid of the hemagglutinating virus of Japan–-liposome, vessels are preferentially transfected after 16-hour preservation (A, x400), and cardiomyocytes are also transfected after subsequent reperfusion (B, x100). No apparent staining is noticed with direct transfer, both after preservation (C, x400) and after reperfusion (D, x100).

 
Posttransplantation cardiac function and serum CPK
There were no significant differences in the recipient hemodynamic variables between the two groups (Table 1). Systolic functional recoveries after 1 hour of reperfusion in terms of LVDP (NF vs SD, 57 ± 5 vs 21 ± 9 mm Hg; p < 0.05) and max dP/dt (1,225 ± 154 vs 235 ± 76 mm Hg/s; p < 0.01) were significantly better in the NF than in the SD group, whereas there was no significant difference in HR between groups (Fig 2).


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  		Table 1. Hemodynamic Parameters of the Heart Transplant Recipient

 


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  		Fig 2. The enhancement of cardiac preservation in terms of the recovery of cardiac performance after 1 hour of reperfusion by transfection of NF{kappa}B decoy. The NF{kappa}B decoy group (NF) showed significantly higher recovery of left ventricular developed pressure (DP) and maximal derivatives of left ventricular pressure (max dP/dt) than did the scrambled decoy group (SD), whereas there was no significant difference in heart rate (HR) between two groups. (* p < 0.05, ** p < 0.01, n = 6 in each group. All values are expressed as mean ± SEM.)

 
Serum CK levels were significantly lower in the NF than in the SD group (3,943 ± 834 vs 7,170 ± 367 IU/L; p < 0.05) (Fig 3).



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  		Fig 3. Serum value of creatine phosphokinase (CPK) after 1 hour of reperfusion. The NF{kappa}B decoy group (NF) showed significantly higher values of serum creatine phosphokinase than did the scrambled decoy group (SD). (* p < 0.05, n = 6 in each group. All values are expressed as mean ± SEM.)

 
Myocardial water content
The myocardial water content in the NF group was significantly lower than in the SD group (78.5% ± 0.6% vs 80.5% ± 0.4%; p < 0.05).

Neutrophil adherence and tissue IL-8
Cytohistochemical staining for neutrophils showed significant blockage of migration or accumulation of neutrophils into the myocardium in the NF group, compared with the SD group (9.7 ± 0.6 vs 14.9 ± 0.9 counts per field with magnification x100; p < 0.01) (Fig 4).



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  		Fig 4. Cytohistochemical staining for neutrophil adherence after reperfusion in the NF{kappa}B decoy group (NF) (A, x200) and in the scrambled decoy group (SD) (B, x200). The number of neutrophils is significantly smaller in the NF than in the SD group (C). (** p < 0.01, n = 6 in each group. All values are expressed as mean ± SEM.)

 
The tissue level of IL-8 was significantly lower in the NF than in the SD group (1,850 ± 161 vs 2,614 ± 18 ng/mg tissue; p < 0.01) (Fig 5).



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  		Fig 5. Tissue IL-8 level measured by ELISA is significantly lower in the NF{kappa}B decoy group (NF) than in the scrambled decoy group (SD). (** p < 0.01, n = 6 in each group. All values are expressed as mean ± SEM.)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
We report here a novel therapeutic strategy to enhance myocardial preservation by attenuating ischemia-reperfusion injury, using an ex vivo transfer of a cis element "decoy" ODN against the NF{kappa}B binding site. The mechanical function of hearts transplanted after 16 hours of hypothermic storage was significantly better preserved in hearts transfected with the NF{kappa}B decoy. In addition, hearts transfected with the NF{kappa}B decoy showed significantly lower levels of serum CK, myocardial water content, and neutrophil adherence accompanied by reduced tissue IL-8 production. These results suggest that NF{kappa}B plays a critical role in myocardial ischemia-reperfusion injury after prolonged preservation, and that blocking NF{kappa}B by gene transfection with a NF{kappa}B decoy is a novel therapy to enhance cardiac preservation by attenuating neutrophil-mediated reperfusion injury.

There have been several reports of ex vivo gene transfection into donor hearts during preservation by perfusion containing an adenovirus [17] or incubation with positive pressure [18]. Using a heterotopic rat heart transplantation model, we have previously reported the efficient gene transfer into cardiomyocytes of donor hearts with the HVJ-liposome method [14]. In the present study, it is noteworthy that delivery of FITC-labeled ODN into endothelial cells was demonstrated during the 16 hours of hypothermic preservation without a subsequent perfusion. This phenomenon may be attributable to HVJ adhering and fusing to endothelial cells under hypothermic condition. This seems to be important when considering the clinical settings of cardiac transplantation, where access to the donor heart is allowed only at the time of harvest.

In myocardial injury after ischemia and reperfusion, endothelial cells have been shown to play an important role because they are anatomically located at the interface of blood and tissue exchange and thus are directly influenced by circulating leukocytes [6–8]. Under hypoxic circumstances endothelial cells appear to be activated to express proinflammatory properties that include the induction of leukocyte-adhesion molecules and cytokines. This hypoxic endothelial cell activation leads to an accumulation of neutrophils in the endothelium and consequently amplifies reperfusion injury. Both IL-8 and ICAM-1 are regulated by the transcription factor NF{kappa}B and both play especially important roles in neutrophil-mediated reperfusion injury. Several studies have shown that the inhibition of NF{kappa}B results in reduction of the enhancement of IL-8 and ICAM-1 expression [9, 10]. In the present study, as well, the blocking of NF{kappa}B resulted in an inhibition of neutrophil adherence accompanied by the reduction of production of IL-8. In addition, our preliminary data showed a tendency for ICAM-1 expression in endothelial cells to be reduced not only after reperfusion but also during the 16-hour hypothermic preservation in the hearts transfected with the NF{kappa}B decoy (data not shown). Although it is still controversial whether the expression of proinflammatory molecules could be upregulated in hypothermic circumstances [19, 20], NF{kappa}B was shown to be translocated to the nucleus at temperatures as low as 17°C, even though active transcription did not take place [20]. Therefore, the transfected NF{kappa}B decoy ODNs might stabilize the phenotype of the microvessel cells so that the expression of proinflammatory molecules is suppressed during hypothermic storage as well as during reperfusion. Further study is still required to clarify the mechanism and effects of the NF{kappa}B decoy on endothelial cell activation under hypothermic conditions.

There have been several strategies devised to enhance cardiac preservation including improvements in preservation solution [2, 16], hypoxic preconditioning [3], and modification of reperfusion [4, 5]. We have previously reported the efficacy of cardioplegia with leukocyte-depleted blood on canine heart grafts preserved for 24 hours, in which we focused on the importance of attenuation of neutrophil-mediated reperfusion injury [4]. An ex vivo gene transfection of the NF{kappa}B decoy supports this concept and seems to be potentially useful because it is technically simple and may act on endothelial activation during hypoxic preservation and after reperfusion.

In conclusion, an ex vivo gene transfection of the cis element "decoy" ODN against NF{kappa}B binding sites attenuated ischemia-reperfusion injury after prolonged heart preservation. As a result, this method appears to be a novel strategy for enhanced myocardial preservation, provided that vectors for gene transfection are proved safe for use in human subjects.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
We thank Akiko Nishimura for excellent technical assistance in preparing HVJ-liposome.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Kaye M.P. The registry of the International Society for Heart and Lung Transplantation: official report 1993. J Heart Lung Transplant 1993;12:541.[Medline]
  2. Gu K., Kin S., Saitoh Y., et al. Cardioprotective effect of nicorandil in histidine-tryptophan-ketoglurate solution during the cold storage of isolated hearts. Transplantation 1996;11:1572-1575.
  3. Engelman D.T., Chen C.Z., Watanabe M., et al. Improved 4-, and 6-hour myocardial preservation by hypoxic preconditioning. Circulation 1995;92(suppl):II417-II422.
  4. Fukushima N., Shirakura R., Nakata S., et al. Study of efficacies of leukocyte-depleted terminal blood cardioplegia in 24-hour preserved hearts. Ann Thorac Surg 1994;58:1651-1656.[Abstract/Free Full Text]
  5. Milliken J.C., Billingsley A.M., Laks H. Modified reperfusate after long-term preservation of the heart. Ann Thorac Surg 1989;47:725-728.[Abstract/Free Full Text]
  6. Szabo G., Fazekas L., Bährle S., et al. Endothelin-A, and -B antagonists protect myocardial, and endothelial function after ischemia/reperfusion in a rat heart transplantation model. Cardiovasc Res 1998;39:683-690.[Medline]
  7. Boyle E.M., Pohman T.H., Cornejo C.J., Verrier E.D. Endothelial cell injury in cardiovascular surgery: ischemia-reperfusion. Ann Thorac Surg 1996;62:1868-1875.[Abstract/Free Full Text]
  8. Murphy C.O., Pan-Chih, Gott J.P., Guyton R.A. Coronary microvascular reactivity after ischemic cold storage and reperfusion. Ann Thorac Surg 1997;63:20-27.[Abstract/Free Full Text]
  9. Morishita R., Sugimoto T., Aoki M., et al. In vivo transfection of cis element "decoy" against nuclear factor-kB binding site prevents myocardial infarction. Nat Med 1997;3:894-899.[Medline]
  10. Sawa Y., Morishita R., Suzuki K., et al. A novel strategy for myocardial protection using in vivo transfection of cis element ‘decoy’ against NF{kappa}B binding site: evidence for a role of NF{kappa}B in ischemia-reperfusion injury. Circulation 1997;96(suppl):II280-II285.
  11. Neish A.S., Williams A.J., Palmer H.J., Whitley M.Z., Collins T. Functional analysis of the human vascular cell adhesion molecule 1 promoter. J Exp Med 1992;176:1583-1593.[Abstract/Free Full Text]
  12. Kaneda Y. Virus(Sendai virus envelopes)-mediated gene transfer. In: Celis J.E., ed. . Cell biology, a laboratory handbook. New York: Academic Press, 1994:50-57.
  13. Morishita R., Gibbons G.H., Ellison K.H., et al. Single intraluminal delivery of antisense cdc 2 kinase oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA 1993;90:8474-8478.[Abstract/Free Full Text]
  14. Sawa Y., Kaneda Y., Bai H.Z., et al. Efficient transfer of oligonucleotides and plasmid DNA into the whole heart thorough the coronary artery. Gene Therapy 1998;5:1472-1480.[Medline]
  15. Ono K., Lindsey E. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg 1969;57:225-229.[Medline]
  16. Baxter K., Howden B.O., Jin X., Jablonski P. Studies in a modified auxiliary abdominal rat heart transplantation model: preservation with colloid-free University of Wisconsin solution. J Heart Transplant 1998;17:532-537.
  17. Gojo S., Niwaya K., Taniguchi S., Nishizaki K., Kitamura S. Gene transfer into the donor heart during cold preservation for heart transplantation. Ann Thorac Surg 1998;65:647-652.[Abstract/Free Full Text]
  18. Mann M.J., Gibbons G.H., Hutchinson H., et al. Pressure-mediated oligonucleotide transfection of rat, and human cardiovascular tissues. Proc Natl Acad Sci USA 1999;96:6411-6416.[Abstract/Free Full Text]
  19. Oz M.C., Liao H., Naka Y., et al. Ischemia-induced interleukin-8 release after human heart transplantation. A potential role for endothelial cells. Circulation 1995;92(suppl):II428-II432.
  20. Haddix T.L., Pohlman T.H., Noel R.F., Sato T.T., Boyle E.M., Verrier E.D. Hypothermia inhibits human E-selectin transcription. J Surg Res 1996;64:176-183.[Medline]

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