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Cardiovascular Research 2006 71(3):527-536; doi:10.1016/j.cardiores.2006.05.021
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Copyright © 2006, European Society of Cardiology

STAT-1 and AP-1 decoy oligonucleotide therapy delays acute rejection and prolongs cardiac allograft survival

Hans Hölschermanna,1, Thomas H.W. Stadlbauera,1, Andreas H. Wagnerb, Horst Fingerhutha, Heidrun Mutha, Song Rongc, Faikah Gülerc, Harald Tillmannsa and Markus Heckerb,*

aUniversity Hospital Giessen, Department of Internal Medicine, Division of Cardiology, Giessen, Germany
bUniversity Hospital Heidelberg, Institute of Physiology and Pathophysiology, Division of Cardiovascular Physiology, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany
cHannover Medical School, Department of Medicine, Division of Nephrology, Hannover, Germany

* Corresponding author. Tel.: +49 6221 544035; fax: +49 641 6221 544038. Email address: hecker{at}physiologie.uni-hd.de

Received 2 June 2005; revised 15 May 2006; accepted 23 May 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Acute myocardial rejection is a cell-mediated process characterized by increased leukocyte recruitment into the graft myocardial tissue. Transcription factors like STAT-1 and AP-1 are critically involved in this process by regulating vascular adhesion molecule expression. The aim of our study was to investigate the effect of decoy oligodeoxynucleotide (dODN) treatment targeting transcription factors AP-1 and STAT-1 on acute cardiac allograft rejection in a rat transplant model.

Methods Wistar–Furth (WF) cardiac allografts were transplanted into Lewis (LEW) rats after perfusion with STAT-1 or AP-1 dODN solution (5 µmol/l), buffer or the corresponding mutant control ODNs. Grafts were harvested and processed for histologic and immunohistochemical evaluation.

Results: As demonstrated by fluorescence dye-labelled dODN, exposure of the grafts to the dODNs during 45 min of warm ischemia resulted in a dominant uptake of naked DNA by the graft endothelium. Treatment with AP-1 and STAT-1 dODNs, but not with vehicle or the control dODNs, significantly prolonged cardiac allograft survival by approximately 40% from 5.6±0.5 days to 7.8±1.3 days and 7.4±0.5 days, respectively (mean±S.D., p<0.01, n=5 in each group). Immunohistochemical examination on days 1, 3 and 6 revealed a marked reduction of infiltrating leukocytes (AP-1 dODN: 85%, STAT-1 dODN: 50%), namely T-cells, in the dODN-perfused grafts at day 3 post transplantation. In addition, as demonstrated by immunohistochemical analysis, endothelial expression of ICAM-1 and VCAM-1 was found to be markedly reduced in dODN-treated grafts.

Conclusion Both AP-1 and STAT-1 dODN treatments suppress graft endothelial adhesion molecule expression, reduce graft infiltration and in turn significantly delay acute rejection. The utilization of dODNs in the cardioplegic solution might be a novel strategy to protect transplanted organs from early damage during transplantation, to preserve organ function and bridge the critical phase after transplantation when standard immunosuppression is not yet completely effective.

KEYWORDS Endothelial function; Gene therapy; Leukocytes; Macrophages; Transplantation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cardiac transplantation has become a treatment of choice for end stage organ failure [1,2]. Despite major improvements in immunosuppressive therapy in the past decade, acute rejection episodes still occur primarily within the first days or weeks after engraftment in cardiac transplant recipients [3–5] and remain an important cause of post-transplant morbidity, mortality and graft loss [1,3–5]. Incidence rates for acute rejection in up to 100% of cardiac allograft recipients receiving standard cyclosporine A (CsA) based immunosuppressive therapy regimens have been reported [6–8]. Acute rejection episodes are known to predispose for development of chronic rejection and chronic allograft dysfunction [9–11] and, therefore, also have a major impact on long-term results after solid organ transplantation.

Mechanistically, acute allograft rejection is the result of an allogeneic, primarily T-cell-dependent response of the recipient's immune system towards the graft [12–14]. Histologically, this response is initiated by a progressive graft infiltration by circulating leukocytes [15,16]. Following the initial process of leukocyte rolling and tethering by P-selectin and integrins, in order for leukocytes to infiltrate the graft, they must adhere to and migrate through the endothelium. This process is regulated by interaction of leukocytes with vascular adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1) [17]. These adhesion molecules are either present constitutively or can be induced after cytokine activation on the graft endothelium. On the molecular level, expression of these adhesion molecules in endothelial cells typically requires activation of the transcription factors nuclear factor kappa B (NF-{kappa}B) and activator protein-1 (AP-1) [18], but, depending on the type of stimulus, namely upon exposure to the prototypic type 1 T-helper cell cytokine interferon-{gamma} (IFN-{gamma}), activation of signal transducer and activator of transcription-1 (STAT-1) also is essential [19]. Targeting these transcription factors may therefore provide a useful approach to influence acute rejection and prolong cardiac allograft survival.

The development of efficient therapeutic strategies to prevent graft rejection, especially during the critical early phase post-transplantation when standard immunosuppressive therapy is not yet fully effective, remains an unsolved clinical problem. Solid organ transplantation thereby is a unique treatment opportunity with respect to the ex vivo situation of the graft during the transplant procedure. Within this time, organ-specific modification of the graft can be performed by adding therapeutic agents to the preservation solution. The ex vivo situation might also offer the possibility for targeting gene expression involved in acute rejection at the level of transcription. In the present study, we investigated whether transfection with decoy oligodeoxynucleotides (dODNs) neutralizing either AP-1 or STAT-1 during the cardiac transplantation procedure attenuates rejection and prolongs cardiac allograft survival of acute rejecting rat cardiac allografts. We show that AP-1 or STAT-1 dODN treatment of the cardiac allograft prior to transplantation decreases expression of the adhesion molecules ICAM-1 and VCAM-1 on the graft endothelium, attenuates the graft infiltration by immune-competent cells, delays acute rejection and prolongs cardiac allograft survival in this rat transplant model.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Animals
Inbred male Lewis (LEW, RT1l) and Wistar–Furth (WF, RT1u) strain rats were purchased from Harlan Winkelmann (Borchen, Germany), Brown Norway (BN, RT1n) and Wistar strain rats were from Charles River (Sulzfeld, Germany). They were housed in the animal care facilities of the University Hospitals Giessen and Heidelberg as well as Hannover Medical School. They were kept under standard temperature, humidity and timed light conditions, and provided with rat chow and water ad libitum. Animals were treated in a humane manner in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985). The transplant procedures were performed when animals attained a body weight of 200 g (kidney transplantation) or 250 g (heart transplantation). LEW rats served as recipients of WF cardiac allografts.

2.2 Cardiac allograft model
The present study used a non-volume-loaded abdominal heterotopic heart transplantation model. Hearts were transplanted to the infrarenal great vessels by standard microvascular techniques in the modified technique of Ono and Lindsey, as previously described [20,21]. Briefly, after the recipient (LEW) and donor rat (WF) had been anaesthetized by i.m. injection of 5% ketamine and 2% xylazine, the abdominal great vessels of the donor and recipients were carefully separated. The donor heart was harvested and flushed with Ringer solution (Na+ 147 mmol/l, K+ 4 mmol/l, Ca2+ 2 mmol/l, Cl– 156 mmol/l) at room temperature. Thereafter, the WF grafts were perfused with 4 ml of AP-1 or STAT-1 consensus dODN (5 µmol/l), AP-1 or STAT-1 mutant dODN (5 µmol/l) or TEN buffer solution (10 mmol/l Tris, 1 mmol/l EDTA and 150 mmol/l NaCl), respectively, and exposed to 45 min of warm ischemia. The treatment groups as well as the control group consisted of five animals in each group for survival analysis. During this time, the donor aorta was anastomosed to the recipient abdominal aorta with 7–0 prolene in an end-to-side technique. The donor pulmonary artery was anastomosed to the recipients' inferior vena cava end-to-side. The tourniquets around the recipient's great vessels were released after a total period of 45 min and reperfusion of the graft started. Within seconds the graft started beating. Thereafter, the abdomen was closed by sutures and graft function was monitored daily by palpation. Rejection was defined as the day of cessation of myocardial contraction. Systemic immunosuppressive therapy was not applied to any animal. For further analysis, grafts from the different experimental groups were harvested at days 1, 3 and 6 after transplantation (n=5 animals for each time point and treatment group).

2.3 Kidney transplantation model
Kidney transplantation was performed as previously described [22]. Briefly, animals were anesthetized and the left donor kidney (BN) attached to a cuff of the aorta. The renal vein and the ureter were removed en bloc. The vascular cuffs were anastomosed to the recipient (LEW) abdominal aorta and vena cava, respectively, below the level of the native renal vessels. Donor and recipient ureter were attached end to end. Both native kidneys of the recipient were removed at the time of transplantation.

2.4 Decoy ODN technique
Double-stranded dODNs were prepared from complementary single-stranded phosphorothiote-bonded ODNs obtained from Eurogentec (Cologne, Germany) by melting at 95 °C for 5 min, followed by a cool-down phase of 3 to 4 h at ambient temperature. The efficiency of the hybridization reaction was verified by 2.5% agarose gel electrophoresis and usually found to exceed 95%. The resulting solution was split into 50 µl aliquots and frozen at – 80 °C until further use. These were defrosted and dissolved in 4 ml Ringer solution yielding a final concentration of 5 µmol/l.

The sequences of the single-stranded ODNs were as follows (underlined letters denote phosphorothiote-bonded bases):

STAT-1 consensus5'-CATGTTATGCATATTCCTGTAAGTG-3' (cf. [23,24])
STAT-1 mutant5'-CATGTTATGCAGACCGTAGTAAGTG-3'
STAT-1 scrambled5'-TGTCATACTCGTACACAC-3'
AP-1 consensus5'-CGCTTGATGACTCAGCCGGAA-3' (cf. [23,25,26])
AP-1 mutant5'-CGCTTGATTACTTAGCCGGAA-3'

As a control, a corresponding volume of TEN buffer was diluted in 4 ml of Ringer solution.

2.5 Fluorescence dye labeling of AP-1 and STAT-1 dODN
DNA uptake in the heart was visualized by perfusion of native hearts with Texas Red labeled AP-1or STAT-1 dODN (n=2), respectively. Following exposure to 45 min of warm ischemia time, hearts were perfused with Ringer solution and embedded in isopentane-precooled OCT compound (Miles, Elkhart, IN, USA), snap-frozen in liquid nitrogen and stored at – 80 °C for fluorescence microscopy examination.

Uptake of the Texas Red labeled nucleic acids was monitored by fluorescence microscopy with a MicroMax 1300Y CCD camera (Printon Instruments, Trenton, NJ, USA) fitted to an Axiovert S100-TV microscope equipped with a 40 x Plan-Neofluar objective (Carl Zeiss, Jena, Germany). Excitation was performed with a xenon lamp and a monochromator (TILL-Photonics, Munich, Germany) set to 580 nm. Emission at 620 nm was recorded with the aid of an appropriate HQ filter set (Chroma Technologies, Brattleboro, VT, USA), and computed by using the MetaMorph software package Version 3.51 (Universal Imaging, West Chester, PA, USA).

2.6 Histology
Allografts were harvested at defined time points (naive, day 1, day 3 and day 6 after transplantation, n=5 for each experimental group and each time point). They were serially sectioned into approximately 2-mm slices, embedded in isopentane-precooled OCT compound, snap frozen in liquid nitrogen and stored at – 80 °C. After fixation in ice-cold acetone for 10 min, cryostat sections (6 µm) were dried and stained with hematoxylin and eosin as described previously [27].

2.7 Immunohistochemical analysis
Two representative slices of the explanted cardiac allografts were embedded in isopentane-precooled OCT compound, snap-frozen in liquid nitrogen and stored at – 80 °C. Dried sections were placed in a 1:750 dilution of rat serum (Sigma, St. Louis, STATE, USA) for 10 min. After rinsing with RPMI 1640 (Gibco, Rockville, STATE, USA), they were incubated with either a mouse monoclonal anti-rat major histocompatibility complex II (MHC II) antibody (dilution 1: 10), an anti-rat ICAM-1 (CD54) antibody (Seikagaku, Tokyo, Japan; dilution 1:75), a goat polyclonal anti-human VCAM-1 (CD106) antibody (Santa Cruz, Biotechnology, Heidelberg, Germany; dilution 1:75), a mouse monoclonal anti-rat monocytes/macrophages antibody (clone ED1; dilution 1:150) or an anti-rat T-cell receptor (TCR) alpha/beta antibody (clone R73; dilution 1:75; both from Serotec, Oxford, England) for 45 min at room temperature. After additional washing steps in Tris buffer, sections were exposed to the secondary antibody, i.e. rabbit anti-mouse immunoglobulin G (IgG) for ICAM-1, ED1, MHC II, R73 (dilution 1:200) or mouse anti-goat IgG for VCAM-1 (dilution 1:300), for 10 min followed, as appropriate, by incubation with a linking antibody (rabbit anti-mouse IgG for VCAM-1, dilution 1:400) for 10 min. Thereafter, an alkaline phosphatase anti-alkaline phosphatase complex (APAAP) (1:50, Dianova, Hamburg, Germany) was added for 20 min. Sections were then developed in New Fuchsin solution (30 min) and counterstained with hematoxylin (Merck, Darmstadt, Germany) for 30 s. Control sections were treated with the linking secondary antibody and APAAP complex only.

Sections from treated and untreated grafts were scored microscopically (0=no staining, 1=weak staining, 2=moderate staining, 3=strong staining) in the post-capillary venules identified by the typical light microscopical signs for ICAM-1 and VCAM-1 staining intensity by two investigators blinded as to the experimental group. The infiltrating cells were quantified by counting the antigen positive-cells in 10 fields of view (magnification 400x) by two independent blinded investigators as described previously [21].

2.8 Preparation and incubation of ring segments of rat thoracic aorta
Aortas (1.5 mm) were isolated from pentobarbitone-anaesthetized male Wistar rats (200–250 g body weight) as described previously [28] and cleaned under sterile conditions of adherent fat and connective tissue. Segments of 5–7 mm in length were placed in 1 ml Waymouth medium containing 10% fetal bovine serum (FBS) and incubated for 16 h in the absence or presence of rat IFN-{gamma} (200 U/ml). In some experiments, segments exposed to IFN-{gamma} were pretreated (4 h) with either the STAT-1 consensus or scrambled control dODN (10 µmol/l). To verify that IFN-{gamma} induction of VCAM-1 expression primarily occurs in the endothelium, one segment was mechanically denuded prior to IFN-{gamma} stimulation.

2.9 Western blot analysis
Following termination of the incubations, protein extracts of the segments were prepared as described previously [28]. They were separated by denaturing 10% polyacrylamide gel electrophoresis in the presence of SDS according to standard protocols and then transferred to a BioTrace polyvinylidene fluoride transfer membrane (Pall Corporation, Roβdorf, Germany). For detection of VCAM-1, the immobilized proteins were consecutively exposed to a polyclonal rabbit anti-VCAM-1 antibody (1:2500 dilution, Santa Cruz Biotechnology), an anti-rabbit IgG-horseradish peroxidase (HRP) conjugate (1:2500 dilution; Sigma-Aldrich, Deisenhofen, Germany) and finally to the ECL Plus Western blotting chemiluminescent reagent (Amersham GE Healthcare, Germany). For chemiluminescence detection, imaging and quantitation of the visualized proteins, the ChemiDoc XRS system and Quantity One analysis software (Bio-Rad, Germany) was used. Loading and transfer of equal amounts of protein in each lane was verified by reprobing the membrane with a monoclonal anti-β-actin antibody from mouse ascites fluid and an anti-mouse IgG-HRP conjugate (both antibodies obtained from Sigma-Aldrich, 1:3000 dilution).

2.10 Gel shift analysis
Gel or electrophoretic mobility shift analyses with the 32P-labeled STAT-1 consensus dODN or the AP-1 consensus dODNs as a probe were performed as described previously [28]. Nuclear extracts were prepared either from isolated vessel branches derived from the BN kidney allografts transplanted into LEW recipients (cf. [28]) or from rat cultured vascular smooth muscle cells which had been cultured as described in detail elsewhere [23,28].

2.11 Statistical analysis
Statistical analysis was performed with the statistics program StatsDirect (Version 2.2.2, Aswell, UK). Results were expressed as mean±S.D., unless otherwise specified. To analyze adhesion molecule expression, 50 post-capillary venules in each section were graded. Graft infiltrating cells were quantified by examination of 10 randomly accessed fields of two sections of each graft for the number of graft infiltrating cells. To analyze statistical differences between experimental and control groups, log-rank test and Mann–Whitney test were applied as indicated, with p-values of <0.05 considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 AP-1 and STAT-1 dODN perfusion prolongs cardiac allograft survival
Untreated control LEW recipients of WF cardiac allografts rejected grafts in an acute fashion with a mean survival time of 5.6±0.5 days. As shown in Fig. 1a, perfusion of the WF donor graft with either AP-1 or STAT-1 dODNs 45 min prior to reperfusion delayed acute cardiac allograft rejection and prolonged allograft survival from 5.6±0.5 days to 7.8±1.3 days (AP-1 dODN) and 7.4±0.5 days (STAT-1 dODN), respectively (mean±S.D., p<0.01, n=5 in each group). Perfusion of the WF donor graft with the corresponding mutant control AP-1 or STAT-1 ODNs had no detectable effect (Fig. 1b).


Figure 1
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  		Fig. 1 Cardiac allograft survival. Mean allograft survival was significantly prolonged in both the AP-1 dODN-treated as well as the STAT-1 dODN-treated allografts when compared to untreated controls (p<0.01, n=5 for each group) or allografts treated with mutant control AP-1 or STAT-1 ODNs (p<0.01, n=5 for each group).

 
3.2 AP-1 and STAT-1 dODN perfusion reduces graft infiltration
In grafts of untreated control recipients, diffuse mononuclear cell infiltration with associated myocyte necrosis and interstitial hemorrhage was seen during the course of acute rejection (Fig. 2). The rejecting untreated grafts showed the typical dense infiltration with ED1-positive monocytes/macrophages and R73-bearing T-cells as well as upregulation of MHC class II molecules. As shown in Fig. 2a (ED1) and Fig. 2b (R73) as well as Table 1, both AP-1 and STAT-1 dODN treatment significantly diminished the number of infiltrating monocytes/macrophages, T-cells, as well as MHC class II-positive cells, respectively.


Figure 2
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  		Fig. 2 Immunohistochemical analysis of graft infiltrating cells. Untreated, acutely rejecting allografts are characterized by an increasing number of infiltrating mononuclear cells from days 1 to 6 post-transplantation. The diffuse inflammatory infiltrate is associated with myocyte necrosis. Immunohistochemistry of days 1, 3 and 6 allografts for ED1-positive monocytes and macrophages (a) or R73-positive T-cells (b) displayed a significantly reduced number of graft infiltrating cells at all time points. Magnification x 400.

 

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  		Table 1 Infiltrating ED1-positive monocytes/macrophages, R73-positive T-cells as well as MHC II-positive cells were quantified by counting the antigen positive cells in 10 fields of view, and sections from treated and untreated grafts were scored microscopically for ICAM-1 and VCAM-1 staining as described in Section 2.7

 
3.3 AP-1 or STAT-1 dODN uptake by endothelial cells
Perfusion of donor hearts with Texas Red-labeled STAT-1 or AP-1 dODNs revealed an exclusive uptake of the administered double-stranded DNA by the endothelium (Fig. 3). No nucleic acid uptake was detectable either within the outer layers of the coronary vessel wall (media, adventitia) or within the myocardial tissue.


Figure 3
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  		Fig. 3 Decoy ODN uptake by endothelial cells. Representative fluorescence photomicrograph of heart tissue sections of native hearts perfused with Texas Red fluorescent dye labeled STAT-1 dODN or AP-1 dODN, respectively. (a,c) Fluorescence image, (b,d) live image. Magnification x 400.

 
3.4 Toxicity of dODN application
Treatment of the donor hearts with either consensus decoy ODN or the mutant control ODNs had no detectable toxic effects, as it did not provoke primary graft failure, alterations in the functional palpation score, nor histological injury compared with the buffer group when analyzed 1 day after the transplantation procedure.

3.5 AP-1 and STAT-1 dODN perfusion inhibits adhesion molecule expression
Untreated acutely rejecting cardiac allografts showed a markedly increased ICAM-1 and VCAM-1 expression in the coronary endothelial cells on days 1 and 3 after engraftment (Fig. 4a and b). As shown in Fig. 4a and b as well as in Table 1, application of either AP-1 or STAT-1 dODN prior to transplantation significantly reduced endothelial ICAM-1 and VCAM-1 expression when compared to the untreated cardiac allografts. Treatment with the mutant control AP-1 or STAT-1 ODNs revealed no such effect.


Figure 4
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  		Fig. 4 Presence of ICAM-1 and VCAM-1 antigen. Immunohistochemical analysis of days 1 and 3 allografts demonstrated a marked attenuation of ICAM-1 and VCAM-1 expression in AP-1 and STAT-1 dODN-treated cardiac allografts. Magnification x 400.

 
3.6 Verification of the mechanism of action and target specificity of the dODNs employed
To verify that the dODNs indeed target expression of the aforementioned pro-inflammatory genes by neutralizing AP-1 or STAT-1, additional experiments were performed with endothelium-intact rat aortic segments. Rat cultured endothelial cells were not employed for this purpose because these cells are difficult to cultivate in sufficient quantity and purity. Exposure of the rat aortic segments to IFN-{gamma} resulted in a significant increase in VCAM-1 protein content which was fully abrogated when the segments had been denuded prior to IFN-{gamma} stimulation (Fig. 5). Moreover, pretreatment with the STAT-1 dODN, but not with a scrambled control ODN, completely blocked IFN-{gamma} stimulated VCAM-1 expression. Basal VCAM-1 expression, approximating 50% of the response to IFN-{gamma}, remained unaffected by either endothelial cell removal or STAT-1 dODN treatment, suggesting that it originated from the vascular smooth muscle cells which express this adhesion molecule constitutively [29].


Figure 5
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  		Fig. 5 Western blot analyses of STAT-1 dODN (10 µmol/l) inhibition of VCAM-1 protein expression induced by IFN-{gamma} in endothelium-intact rat aortic segments. Representative Western blot analysis and statistical summary (n=5–10, *p<0.01 vs. stimulated control, #p<0.01 vs. scrambled control ODN (STAT-1 scr)). For experimental details, refer to the text.

 
To demonstrate that the dODNs were indeed specific for their target transcription factor, nuclear extracts were prepared from isolated vessel branches of BN kidney allografts transplanted into LEW recipients. Only treatment with the STAT-1 dODN resulted in a diminished nuclear translocation of STAT-1 when compared to the corresponding mutant control ODN or the AP-1 dODN 24 h post-transplantation (Fig. 6a). This specific neutralizing effect of the dODNs appears to be attained at the femtomole level inside the target cell (Fig. 6b) and, as shown for the AP-1 dODN in rat cultured vascular smooth muscle cells, only a relatively small (i.e., 20-fold) excess of the dODNs over the endogenous transcription factor binding sites may suffice to fully neutralize the target transcription factor.


Figure 6
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  		Fig. 6 (a) Representative gel shift analyses demonstrating the effects of perfusion followed by 30 min warm ischemia of kidney allografts with the STAT-1 dODN, the corresponding mutant control ODN or the AP-1 dODN (10 µmol/l each) on the nuclear translocation of STAT-1 (marked by arrows) in isolated vessel branches derived from the allografts 24 h post-transplantation. In this fully allogeneic (i.e., RT1n to RT1l) strong rejecting rat kidney transplant model, the STAT-1 dODN also exerted a significant protective effect. (b) Representative gel shift analysis demonstrating the efficacy and specificity of the AP-1 dODN (c) in a cell-free system, i.e. a nuclear extract of rat cultured vascular smooth muscle cells (m, mutant control ODN).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The major findings of the present study are that transfection of cardiac allografts during the transplant procedure with dODNs targeting either AP-1 or STAT-1 transcription factors (i) suppresses ICAM-1 and VCAM-1 expression in the early phase of rejection; (ii) diminishes the inflammatory response by reducing cellular infiltration with immune-competent cells, such as T-cells, monocytes/macrophages as well as MHC class II-positive cells at advanced time points of graft rejection; and (iii) significantly prolongs cardiac allograft survival without systemic administration of additional immunosuppressive therapy.

Different specific and nonspecific immunosuppressive strategies have been applied to abrogate acute rejection and render long-term graft acceptance following cardiac transplantation. Though some of these immunosuppressive strategies induce long-term graft survival, they do not prevent acute rejection episodes, especially during the critical early phase post-transplantation when standard immunosuppressive therapy is not yet fully effective. Direct targeting of genes involved in acute rejection at the level of transcription might emerge as useful strategy for interfering with the cellular events leading to transplant rejection.

Previous work has shown that activation of distinct transcription factors as well as cellular activation pathways can effectively be blocked by decoy oligonucleotides (dODNs), providing a potential therapeutic approach for treatment of inflammatory diseases such as ischemia–reperfusion injury [30,31], cardiac allograft rejection [32–34] as well as restenosis post-angioplasty [26]. In the present study, we found that cardiac allografts treated with dODNs mimicking the consensus binding site of the transcription factors AP-1 and STAT-1 had a significantly longer survival than untreated grafts. The observed delayed myocardial rejection under allograft dODN treatment thereby was shown to result–at least in part–from attenuating the transcriptional activation of endothelial adhesion molecules required for binding, transendothelial migration and activation of T-cells and antigen-presenting cells. Even though NF-{kappa}B appeared to be an equally attractive target for this approach [31,35], we did not test the corresponding decoy oligonucleotides here due to concerns of a possible pro-apoptotic effect on the coronary endothelial cells [36,37]. The following observations suggest that the graft endothelium is the major site of action of the dODNs therapeutic effect and that the effect is sequence-specific: (i) As demonstrated by immunofluorescence labeling of dODNs, ex vivo exposure of grafts for 45 min to warm ischemia resulted in an exclusive uptake of the dODNs by the graft endothelial cells but neither by cells of the outer layers of the coronary vessel wall nor myocardial cells. (ii) Application of AP-1 and STAT-1 consensus dODNs, respectively, but not the corresponding mutant control ODNs, significantly attenuated the expression of the adhesion molecules ICAM-1 and VCAM-1 in graft coronary endothelial cells. (iii) Inhibition of adhesion molecule expression on the graft vascular endothelium by dODN application was accompanied by a marked reduction of leukocyte infiltration into the graft.

Our findings confirm clinical studies demonstrating a consistent increase in ICAM-1 and VCAM-1 on endothelium from arterioles, venules, capillaries and endocardium in biopsies during acute rejection episodes in human transplant recipients [38]. The observation that simultaneous inhibition of ICAM-1 and VCAM-1 upregulation significantly attenuates graft mononuclear infiltration is in line with previous work performed in rat transplant models [21,39].

The local administration of dODNs during preservation solution induced arrest within the transplant procedure–without use of hyperbaric transfection methods, viral vectors, lipid formulations or exposure to other adjunctive, potentially hazardous substances or procedures–proved to be safe as it had no detectable immediate toxic effects on the transfected allografts. In contrast to studies in which transfection with lipid vectors, plasmids, viruses or pressurization was shown to primarily target myocardial cells [33,40,41], we found that pure ex vivo perfusion of the cardiac allograft efficiently transfected "naked" DNA exclusively into the graft's endothelial cells, presumably through an energy-dependent carrier-mediated transport process [42]. Reflecting the critical role that the activated graft endothelium plays in transplant pathoimmunology, a selective targeting of the graft vascular endothelial compartment by preventing endothelial activation appears to be an attractive and pathophysiologically reasonable approach to influence allograft rejection.

In conclusion, single application of "naked" AP-1 or STAT-1 dODNs during the transplantation procedure attenuated rejection in this fully allogeneic (i.e., RT1u to RT1l), strong rejecting rat cardiac transplant model presumably by inhibiting leukocyte–endothelial interaction and, as a consequence, reducing mononuclear cell infiltration after cardiac transplantation. Our findings that AP-1 and STAT-1 inhibition not only prolonged survival but also reduced myocardial inflammation and myocardial tissue damage suggest that this strategy might help to preserve ventricular function and enhance salvage of cardiac muscle cells in the very early phase after heart transplantation. Ex vivo perfusion of cardiac allograft with dODNs thus may serve as a simple, safe, efficient, comfortable and highly targeted method, which can help to bridge the critical phase after transplantation when standard immunosuppression is not yet completely effective.


   Acknowledgement
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft SFB 547/A5 and SFB 405/B17.


   Notes
 
1 Both authors contributed equally to this work. Back

Time for primary review 27 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Taylor D.O., Edwards L.B., Boucek M.M., Trulock E.P., Keck B.M., Hertz M.I. The Registry of the International Society for Heart and Lung Transplantation: twenty-first official adult heart transplant report. J Heart Lung Transplant (2004) 23:796–803.[CrossRef][ISI][Medline]
  2. Deng M.C., De Meester J.M., Smits J.M., Heinecke J., Scheld H.H. Effect of receiving a heart transplant: analysis of a national cohort entered on to a waiting list, stratified by heart failure severity. Comparative Outcome and Clinical Profiles in Transplantation (COCPIT) Study Group. BMJ (2000) 321:540–545.[Abstract/Free Full Text]
  3. Kobashigawa J.A., Kirklin J.K., Naftel D.C., Bourge R.C., Ventura H.O., Mohanty P.K., et al. Pretransplantation risk factors for acute rejection after heart transplantation: a multiinstitutional study, The Transplant Cardiologists Research Database Group. J Heart Lung Transplant (1993) 12:355–366.[ISI][Medline]
  4. Hauptman P.J., Nakagawa T., Tanaka H., Libby P. Acute rejection: culprit or coincidence in the pathogenesis of cardiac graft vascular disease? J Heart Lung Transplant (1995) 14:S173–S180.[ISI][Medline]
  5. Costanzo M.R., Naftel D.C., Pritzker M.R., Heilman J.K. Jr., Boehmer J.P., Brozena S.C., et al. Heart transplant coronary artery disease detected by coronary angiography: a multiinstitutional study of preoperative donor and recipient risk factors. Cardiac Transplant Research Database. J Heart Lung Transplant (1998) 17:744–753.[ISI][Medline]
  6. Reichart B., Meiser B., Vigano M., Rinaldi M., Martinelli L., Yacoub M., et al. European Multicenter Tacrolimus (FK506) Heart Pilot Study: one-year results–European Tacrolimus Multicenter Heart Study Group. J Heart Lung Transplant (1998) 17:775–781.[ISI][Medline]
  7. Meiser B.M., Überfuhr P., Fuchs A., Schmidt D., Pfeiffer M., Paulus D., et al. Single-center randomized trial comparing tacrolimus (FK506) and cyclosporine in the prevention of acute myocardial rejection. J Heart Lung Transplant (1998) 17:782–788.[ISI][Medline]
  8. Meiser B.M., Pfeiffer M., Schmidt D., Reichenspurner H., Überfuhr P., Paulus D., et al. Combination therapy with tacrolimus and mycophenolate mofetil following cardiac transplantation: importance of mycophenolic acid therapeutic drug monitoring. J Heart Lung Transplant (1999) 18:143–149.[CrossRef][ISI][Medline]
  9. Tullius S.G., Tilney N.L. Both alloantigen-dependent and -independent factors influence chronic allograft rejection. Transplantation (1995) 59:13–18.
  10. Weis M., von Scheidt W. Cardiac allograft vasculopathy: a review. Circulation (1997) 96:2069–2077.[Abstract/Free Full Text]
  11. Orbaek Andersen H. Heart allograft vascular disease: an obliterative vascular disease in transplanted hearts. Atherosclerosis (1999) 142:243–263.[CrossRef][ISI][Medline]
  12. Tilney N.L., Kupiec-Weglinski J.W. The biology of acute transplant rejection. Ann Surg (1991) 214:98–106.[ISI][Medline]
  13. Strehlau J., Pavlakis M., Lipman M., Shapiro M., Vasconcellos L., Harmon W., et al. Quantitative detection of immune activation transcripts as a diagnostic tool in kidney transplantation. Proc Natl Acad Sci U S A (1997) 94:695–700.[Abstract/Free Full Text]
  14. Pattison J.M., Krensky A.M. New insights into mechanisms of allograft rejection. Am J Med Sci (1997) 313:257–263. [14].[CrossRef][ISI][Medline]
  15. Mason D.W., Morris P.J. Effector mechanisms in allograft rejection. Annu Rev Immunol (1986) 4:119–145.[CrossRef][ISI][Medline]
  16. Barry W.H. Mechanisms of immune-mediated myocyte injury. Circulation (1994) 89:2421–2432.[Abstract/Free Full Text]
  17. Carlos T.M., Harlan J.M. Leukocyte–endothelial adhesion molecules. Blood (1994) 84:2068–2101.[Abstract/Free Full Text]
  18. Roebuck K.A., Finnegan A. Regulation of intercellular adhesion molecule-1 (CD54) gene expression. J Leukoc Biol (1999) 66:876–888. [18].[Abstract]
  19. Jaruga B., Hong F., Kim W.H., Gao B. IFN-gamma/STAT1 acts as a proinflammatory signal in T cell-mediated hepatitis via induction of multiple chemokines and adhesion molecules: a critical role of IRF-1. Am J Physiol Gastrointest Liver Physiol (2004) 287:G1044–G1052.[Abstract/Free Full Text]
  20. Ono KLindsey E.S. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg (1969) 57:225–229.[ISI][Medline]
  21. Fingerhuth H., Hölschermann H., Grimm H., Tillmanns H., Haberbosch W., Braun-Dullaeus R.C., et al. 3-Deazaadenosine prevents leukocyte invasion by suppression of adhesion molecule expression during acute cardiac allograft rejection: involvement of apoptotic cell death. J Heart Lung Transplant (2004) 23:970–978.[CrossRef][ISI][Medline]
  22. Noris M., Azzollini N., Mister M., Pezzotta A., Piccinini G., Casiraghi F., et al. Peripheral donor leukocytes prolong survival of rat renal allografts. Kidney Int (1999) 56:1101–1112.[CrossRef][ISI][Medline]
  23. Krzesz R., Wagner A.H., Cattaruzza M., Hecker M. Cytokine-inducible CD40 gene expression in vascular smooth muscle cells is mediated by nuclear factor kappaB and signal transducer and activation of transcription-1. FEBS Lett (1999) 453:191–196.[CrossRef][ISI][Medline]
  24. Wagner A.H., Gebauer M., Pollok-Kopp B., Hecker M. Cytokine-inducible CD40 expression in human endothelial cells is mediated by interferon regulatory factor-1. Blood (2002) 99:520–525.[Abstract/Free Full Text]
  25. Lauth M., Wagner A.H., Cattaruzza M., Orzechowski H.D., Paul M., Hecker M. Transcriptional control of deformation-induced preproendothelin-1 gene expression in endothelial cells. J Mol Med (2000) 78:441–450.[CrossRef][ISI][Medline]
  26. Buchwald A.B., Wagner A.H., Webel C., Hecker M. Decoy oligodeoxynucleotide against activator protein-1 reduces neointimal proliferation after coronary angioplasty in hypercholesterolemic minipigs. J Am Coll Cardiol (2002) 39:732–738.[Abstract/Free Full Text]
  27. Adams D.H., Tilney N.L., Collins J.J. Jr., Karnovsky M.J. Experimental graft arteriosclerosis: I. The Lewis-to-F-344 allograft model. Transplantation (1992) 53:1115–1119.[ISI][Medline]
  28. Wagner A.H., Schwabe O., Hecker M. Atorvastatin inhibition of cytokine-inducible nitric oxide synthase expression in native endothelial cells in situ. Br J Pharmacol (2002) 136:143–149. [39].[CrossRef][ISI][Medline]
  29. Cattaruzza M., Schäfer K., Hecker M. Cytokine-induced down-regulation of zfm1/splicing factor-1 promotes smooth muscle cell proliferation. J Biol Chem (2002) 277:6582–6589.[Abstract/Free Full Text]
  30. Dragun D., Lukitsch I., Tullius S.G., Qun Y., Park J.K., Schneider W., et al. Inhibition of intercellular adhesion molecule-1 with antisense deoxynucleotides prolongs renal isograft survival in the rat. Kidney Int (1998) 54:2113–2122.[CrossRef][ISI][Medline]
  31. Vos I.H., Govers R., Grone H.J., Kleij L., Schurink M., De Weger R.A., et al. NFkappaB decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts. FASEB J (2000) 14:815–822.[Abstract/Free Full Text]
  32. Mann M.J., Gibbons G.H., Hutchinson H., Poston R.S., Hoyt E.G., Robbins R.C., et al. Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues. Proc Natl Acad Sci U S A (1999) 6:6411–6416.
  33. Poston R.S., Mann M.J., Hoyt E.G., Ennen M., Dzau V.J., Robbins R.C. Antisense oligodeoxynucleotides prevent acute cardiac allograft rejection via a novel, nontoxic, highly efficient transfection method. Transplantation (1999) 68:825–832.[CrossRef][ISI][Medline]
  34. Yamaguchi A., Miniati D.N., Hirata K., Hoyt E.G., Robbins R.C. Ex vivo blockade of endothelin-1 inhibits graft coronary artery disease in a rodent cardiac allograft model. J Heart Lung Transplant (2002) 21:417–424.[CrossRef][ISI][Medline]
  35. Ohmori K., Takeda S., Miyoshi S., Minami M., Nakane S., Ohta M., et al. Attenuation of lung injury in allograft rejection using NF-kappaB decoy transfection-novel strategy for use in lung transplantation. Eur J Cardiothorac Surg (2005) 27:23–27.[Abstract/Free Full Text]
  36. Li X., Stark G.R. NFkappaB-dependent signaling pathways. Exp Hematol (2002) 30:285–296.[CrossRef][ISI][Medline]
  37. Feeley B.T., Miniati D.N., Park A.K., Hoyt E.G., Robbins R.C. Nuclear factor-kappaB transcription factor decoy treatment inhibits graft coronary artery disease after cardiac transplantation in rodents. Transplantation (2000) 70:1560–1568.[CrossRef][ISI][Medline]
  38. Taylor P.M., Rose M.L., Yacoub M.H., Pigott R. Induction of vascular adhesion molecules during rejection of human cardiac allografts. Transplantation (1992) 54:451–457.[ISI][Medline]
  39. Deuse T., Schrepfer S., Schafer H., Koch-Nolte F., Schwedhelm E., Böger R.H., et al. FK778 attenuates lymphocyte–endothelium interaction after cardiac transplantation: in vivo and in vitro studies. Transplantation (2004) 78:71–77.[ISI][Medline]
  40. Qin L., Chavin K.D., Ding Y., Favaro J.P., Woodward J.E., Lin J., et al. Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Immunosuppression with TGF-beta 1 or vIL-10. Transplantation (1995) 59:809–816.[ISI][Medline]
  41. Dalesandro J., Akimoto H., Gorman C.M., McDonald T.O., Thomas R., Liggitt H.D., et al. Gene therapy for donor hearts: ex vivo liposome-mediated transfection. J Thorac Cardiovasc Surg (1996) 111:416–421.[Abstract/Free Full Text]
  42. Wagner A.H., Fleischer J., Burkhardt B.C., Burkhardt G., Hecker M. Mechanism of DNA oligonucleotide uptake by human endothelial cells. Pflügers Arch Eur J Physiol (2004) 447(Suppl.1):S30. [abstract].

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T. H.W. Stadlbauer, A. H. Wagner, H. Holschermann, S. Fiedel, H. Fingerhuth, H. Tillmanns, R. M. Bohle, and M. Hecker
AP-1 and STAT-1 decoy oligodeoxynucleotides attenuate transplant vasculopathy in rat cardiac allografts
Cardiovasc Res, June 13, 2008; (2008) cvn135v2.
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