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Ann Thorac Surg 1999;68:119-124
© 1999 The Society of Thoracic Surgeons

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Original Articles

Pressure delivery of AS-ICAM-1 ODN with LFA-1 mAb reduces reperfusion injury in cardiac allografts

^a Department of Cardiothoracic Surgery, Stanford University Medical Center, Stanford, California, USA

Address reprint requests to Dr Robbins, Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA 94305-5407
e-mail: robbins{at}leland.stanford.edu

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. The goal of this study is to determine the effects of ex vivo hyperbaric pressure administration of AS-ICAM-1 ODN and systemic anti-LFA-1 mAb treatment on reperfusion injury in the rat cardiac allograft model.

Methods. A PVG to ACI functional heterotopic rat heart model was used. Donor hearts were treated with either saline or AS-ICAM-1 ODN and 5 atm of hyperbaric pressure for 45 minutes. Anti-LFA-1 mAb was administered systemically prior to reperfusion of the allograft. Allografts were procured 24 hours after transplantation for assessment of reperfusion injury or 72 hours to determine ICAM-1 protein expression.

Results. Ex vivo administration of AS-ICAM-1 ODN led to decreases in percentage wet weight (77.1 ± 0.83% vs 78.7 ± 1.0%, p< 0.05), myeloperoxidase activity (3.14 ± 0.72 vs 4.07 ± 0.59, p < 0.05), contraction band necrosis (6.4 ± 6.47% vs 21.1 ± 7.43%, p< 0.01), and ICAM-1 protein expression determined by immunohistochemistry compared to saline controls. Treatment with anti-LFA-1 mAb resulted in decreases in wet weight ratio (76.7 ± 0.63%,p < 0.05 vs saline), myeloperoxidase activity (3.58 ± 0.39,p < 0.05 vs saline) and contraction band necrosis (11.8 ± 3.56%, p < 0.05 vs saline). Combination of pressure administration of AS-ICAM-1 ODN and anti-LFA-1 mAb decreased wet weight ratios (77.1 ± 0.93%, p < 0.05 vs saline), myeloperoxidase activity (2.88 ± 0.44, p < 0.01 vs saline), and contraction band necrosis (6.75 ± 5.67%,p < 0.05 vs saline).

Conclusions. Ex vivo pressure mediated delivery of AS-ICAM-1 ODN decreases ICAM-1 protein expression, reduces reperfusion injury in rodent cardiac allografts, and is more effective than anti-LFA-1 mAb treatment alone.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
Antisense oligodeoxynucleotide (AS-ODN) treatment has been used as an effective method of gene therapy in many applications [1, 2]. Current vectors such as liposomes and viruses, however, have significant disadvantages, including low transfection efficiency, lack of tissue specificity, and immunogenicity [3]. Hyperbaric pressure has been shown to be a safe and effective vector to deliver AS-ODN into both cardiac allografts and vein grafts [4–6]. Solid organ transplantation allows for selective ex vivo treatment with AS-ODN and thus avoids the complications associated with systemic administration.

AS-ODN hybridize to specific mRNA and inhibit gene expression by Watson-Crick base pairing [1, 7]. Once the mRNA is bound by the AS-ODN, translational arrest occurs and the mRNA is rapidly degraded by RNAses [8]. A phosphothiolate backbone on the AS-ODN substitutes a sulfur for an oxygen atom on the DNA backbone, and allows the AS-ODN molecule to resist nuclease degradation in vivo [9].

During periods of reperfusion following ischemia, myocardial endothelial cells express specific leukocyte attraction molecules including intercellular adhesion molecule–1 (ICAM-1) [2, 10]. Reperfusion injury is characterized by two separate phases. First, oxygen free radicals released from ischemic tissue, as well as invading neutrophils, produce local necrosis and continued upregulation of endothelial cell surface proteins. Second, increased expression of leukocyte attraction molecules then promotes binding, diapedesis, and infiltration of neutrophils into the damaged tissue [11]. The end result of reperfusion injury in cardiac allografts is regions of necrosis and dykinesis [12].

The role of ICAM-1 in reperfusion injury and neutrophil attraction has been well documented [11, 12]. ICAM-1 is an inducible protein that is expressed in high levels on vascular endothelium after reperfusion injury [12]. It is solely transcriptionally upregulated, suggesting that blockade of protein translation could lead to blockade of ICAM-1 expression. The interaction of ICAM-1 with its ligand LFA-1, which is present on neutrophils and other leukocytes, is an important component of numerous immunologic processes [13]. It enhances neutrophil activity, T cell interaction with antigen presenting cells, T cell activation of B cells, and adhesion and diapedesis of leukocytes into the myocardium [11, 14, 15]. Enhanced allograft survival has been achieved with monoclonal antibody blockade of LFA-1 and ICAM-1 in rodent models [16]. Moreover, mice that are deficient in ICAM-1 have been shown to be protected against ischemic injury [17, 18]. Finally, antisense oligonucleotides to ICAM-1 have been shown to bind ICAM-1 mRNA in a sequence specific manner and block protein expression [19]. Use of a phosphothiolated antisense sequence to limit degradation by cellular nucleases also increases the efficiency of antisense gene therapy [1, 9, 20].

In previous studies, we optimized hyperbaric pressure as a potent vector for ex vivo myocardial delivery of AS-ODN [4]. The present study was done to assess the effects of ICAM-1 and LFA-1 blockade on reperfusion injury in a functional heterotopic rodent heart model.

	Material and methods

Top Abstract Introduction Material and methods Results Comment References

 
Adult male PVG (RT1a) rats weighing between 250–325g were used as donors. Adult male ACI (RT1c) rats (285–350g) were used as recipients. Both strains were obtained from Harlan Sprague Dawley, Indianapolis, IN. Animals were maintained at the animal care facilities of the Department of Cardiothoracic Surgery, Stanford University Medical Center, Stanford, CA, under standard temperature, humidity, and 12-hour light/dark cycles. Food and water were provided ad libitum. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86–23, revised 1985).

Anesthesia
Prior to organ procurement or transplantation, animals were anesthetized by inhalation of 1 to 2% methoxyfluorane followed by an intraperitoneal injection of pentobarbital (50 mg/kg for PVG, 40 mg/kg for ACI). During anesthesia, heart rate, respiratory rate, and temperature were closely monitored.

Procurement and administration of pressure
Native hearts were procured from donor (PVG) rats following systemic heparinization using a modification of the technique described by Ono and Lindsay [21]. Three cc of Stanford cardioplegia solution was administered proximal to an aortic cross-clamp followed by infusion of either 1.2 cc of 80 µmol/L AS-ICAM-1 ODN or saline. The right atrial appendage was opened with a small incision and an ASD was made with a standard 2.5-mm cardiac punch. The tricuspid valve was made incompetent by ablation of one of the leaflets via the opening in the atrial appendage. The pulmonary artery and veins were then tied off separately and the heart was removed from the thoracic cavity.

Hearts were placed in a pressure chamber at 4°C after which the pressure was increased using air at a rate of 4 atm per minute to a total of 5 atm. Pressure was decreased at a rate of 2 atm over 5 minutes. Total incubation time was measured from induction to complete withdrawal of pressure.

Transplantation
Normal heterotopic heart transplants were performed in the conventional manner with the donor aorta anastomosed to the recipient abdominal aorta and the donor pulmonary artery anastomosed to the recipient inferior vena cava [21]. In the functional heart configuration, an end-to-side anastomosis of donor aorta to recipient abdominal aorta was performed similar to the conventional heterotopic method. A venotomy of approximately 7 to 8 mm was made to accommodate the lengthy incision of the donor right atrial appendage. The right atrial appendage was trimmed and anastomosed to the recipient inferior vena cavas with continuous running suture. Animals were fully recovered after a maximum of three hours. Graft function was assessed by daily palpation.

AS-ICAM-1 ODN and anti-LFA antibody
The Protein, Amino acid, and Nucleotide (PAN) facility at Stanford University, Stanford, CA, synthesized the AS-ODN with a phosphothiolate backbone and dissolved in phosphate buffered saline (PBS) at a concentration of 80 µmol/L. The AS-ICAM-1 ODN had the following sequence: 5’-TGCATCCCCAGGCCACTGTC-3’ [6].

The LFA-1 monoclonal antibody was harvested from abdominal ascites using the E-Z-Sep IgG purification kit (Middlesex Sciences, Inc, MA). Biologic effect and purity of the antibody was ascertained by measuring its binding affinity to purified LFA-1 protein on western blot analysis.

Experimental groups
Six treatment groups were tested with a minimum of five rats in each group (Table 1 ). In addition, 5 PVG rats were sacrificed and hearts procured for a baseline control of biochemical and histologic data (group 1). Group 2 animals received no treatment or pressure incubation, and served as a negative control. Hearts from the other 5 groups were treated with 5 atm hyperbaric pressure for 45 minutes. Group 3 hearts received 1.2 cc saline and were transplanted with the standard heterotopic technique. Hearts from groups 4 to 7 were transplanted with the functional heart configuration.

[(Wet Weight-Dry Weight)/Wet Weight] x 100

Sections taken for histologic analysis were fixed in 10% formalin and embedded in paraffin. Standard hematoxylin and eosin as well as Trichrome staining were performed on 5 micron paraffin sections (Histotec Laboratories, Hayward, CA) Contraction band necrosis was assessed with computer assisted analysis (C-imaging Systems, Cranberry Twp, NJ) to determine the total area of myocardium with contraction bands as a percentage of the total area on the section. Six sections were analyzed for each heart (2 each from right ventricle, left ventricle, and septum).

Immunohistochemical analysis was performed on allografts procured 72 hours after transplantation for maximal ICAM-1 protein expression. Sections were procured and snap frozen in liquid nitrogen. A primary mouse anti-rat monoclonal anti-ICAM-1 antibody was used (Serotec Ltd, United Kingdom) followed by staining with secondary goat anti-mouse antibody with a horse radish peroxidase enzyme kit (Zymed, South San Francisco, CA). Expression of ICAM-1 protein was assessed in a semiquantitative manner, from 0 (no expression) to 3 (entire field shows expression) on three sections (left ventricle, right ventricle, septum). All histologic analyses were performed by a researcher blinded to treatment groups.

Statistical analysis
Values are indicated as a mean ± standard deviation. Groups were compared using a student’s t test. Statistical significance was defined as p < 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
Toxicity of pressure and functional heart model
Donor hearts that underwent the standard heterotopic heart transplant (group 2) showed increases in wet weight ratio compared to normal PVG hearts (group 1) (78.1± 1.1% vs 75.4 ± 0.8%, respectively; p < 0.05). Myeloperoxidase activity (2.42 ± 0.31 vs 2.01 ± 0.28, respectively; p< 0.05), and contraction band necrosis (14.8 ± 3.35% vs 6.5 ± 4.1%, respectively; p < 0.01) were also significantly higher.

Hearts treated with 1.3 cc saline at 5 atm of pressure (group 3) had no significant difference in percent wet weight and contraction band necrosis. However, there was a significant increase in myeloperoxidase activity in hearts treated with hyperbaric pressure (Table 2 ).

Treatment with AS-ICAM-1 ODN and anti LFA-1 mAb
The effects of ex vivo pressure treatment with AS-ICAM-1, anti-LFA-1 mAb treatment, and a combination of the two are shown in Figures 1 and 2. Hearts treated with 80 µmol/L AS-ICAM-1 ODN (group 5) demonstrated no significant difference in heart score compared to the saline controls, but there was a decrease in percentage wet weight (77.1 ± 0.83%,p < 0.05 vs group 4), myeloperoxidase activity (3.14 ± 0.72, p< 0.05 vs group 4), and contraction band necrosis (6.4 ± 4.96%, p< 0.01 vs group 4). Immunohistochemistry analysis demonstrated that ICAM-1 protein expression in AS-ICAM-1 ODN treated allografts decreased compared to saline controls (0.92 ± 0.31, p< 0.001 vs 2.11 ± 0.34, saline group 4) (Fig 3 ).

View larger version (33K): [in this window] [in a new window]   Fig 1. Histograms depicting the effects of ex vivo hyperbaric pressure administration of AS-ICAM-1 ODN and systemic anti-LFA-1 mAb on reperfusion injury. (A) Percent wet weight analysis measuring cardiac edema shows a decrease with treated groups over saline controls. (B) Myeloperoxidase activity assaying for neutrophil infiltration in the allograft demonstrates that combination treatment had the greatest blockade of neutrophil infiltration. (C) Contraction band necrosis percentage to measure histologic damage shows a significant decrease in allograft damage in all treated groups. (D) Heart scores demonstrating that all groups had satisfactory allograft function. * = p< 0.05 compared to saline controls.

View larger version (145K): [in this window] [in a new window]   Fig 2. Photomicrographs of allograft sections taken 24 hours after transplantation. Saline controls (A) show significant contraction band necrosis (arrows). Treatment with AS-ICAM-1 ODN (B), anti-LFA-1 mAb (C), or both (D), achieved a decrease in contraction band necrosis.

View larger version (45K): [in this window] [in a new window]   Fig 3. Immunohistochemistry analysis of ICAM-1 protein expression. ICAM-1 protein stains brown in the sections. (A) Saline control shows high levels of expression of ICAM-1, especially around vessels (arrow). (B) Treatment with AS-ICAM-1 ODN decreases ICAM-1 expression. (C) Combination of AS-ICAM-1 ODN and LFA-1 mAb shows similar ICAM-1 expression as (B).

Combination treatment of 80 µmol/L AS-ICAM-1 ODN at 5 atm for 45 minutes and administration of anti-LFA-1 mAb (group 7) decreased percent wet weight (77.1 ± 0.93%, p< 0.05 vs group 4) and contraction band necrosis (6.75 ± 5.6%, p< 0.01 vs group 4) to levels similar to treatment with hyperbaric AS-ICAM-1 ODN. However, there was a significant decrease in myeloperoxidase activity over all other treatment groups with this regimen (2.88 ± 0.44, p< 0.01 vs group 4). Immunohistochemistry analysis showed no significant difference in ICAM-1 protein expression between combination treatment and pressure mediated delivery of AS-ICAM-1 ODN alone (1.04 ± 0.31, p= not significant vs group 5) (Figure 3).

In all cases, the mean heart scores indicated that all grafts were beating well after 24 hours. The groups had a mean heart score ranging from 3 (group 2) to 4 (group 6), with no significant difference between them.

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
It has been shown that treatment of cardiac allograft recipients with anti-LFA-1 mAb can block acute rejection and in some cases induce tolerance [23]. The present study analyzes the effects of ex vivo myocardial ICAM-1 blockade alone or in combination with recipient systemic blockade of LFA-1 on reperfusion injury in a functional cardiac allograft.

Initially, the level of reperfusion injury in the standard cardiac allograft model was established. Donor hearts showed an increase in cardiac edema, myeloperoxidase activity, and contraction band necrosis when compared to unmanipulated native PVG hearts.

Next, the effects of increased hyperbaric pressure on reperfusion injury were examined. Increasing from ambient pressure to 5 atm for 45 minutes did not increase cardiac edema or contraction band necrosis; however, there was an increase in myeloperoxidase activity. This suggests that hyperbaric pressure may cause limited neutrophil associated myocardial damage. This is consistent with previous data that has shown hyperbaric pressure to be relatively nontoxic to cardiac allografts [4]. Finally, there was no difference in toxicity between groups that underwent the normal and functional heterotopic heart transplants.

Treatment with AS-ICAM-1 ODN at 5 atm of pressure leads to a decrease in percentage wet weight, myeloperoxidase activity, and contraction band necrosis. ICAM-1 is normally expressed in high levels after periods of ischemia followed by reperfusion [2]. Hearts treated with hyperbaric induction of AS-ICAM-1 ODN had biochemical assay scores near to the baselines for myeloperoxidase activity and contraction band necrosis. Our previous data demonstrates that hyperbaric induction of AS-ICAM-1 ODN into the donor heart is able to decrease ICAM-1 protein expression on the graft and thereby decrease reperfusion injury [6]. Immunohistochemical analysis in this study demonstrated that pressure mediated treatment of allografts with AS-ICAM-1 ODN lowers protein expression on the myocardium.

Studies have shown that LFA-1 blockade will decrease reperfusion injury and acute rejection [23]. We also found treatment of the recipient with anti-LFA-1 mAb reduces reperfusion injury, as suggested by the fact that there was a decrease in wet weight, myeloperoxidase activity, and contraction band necrosis in animals when compared to control animals. However, the decrease in myeloperoxidase activity and contraction band necrosis was not as low as the blockade with AS-ICAM-1 ODN administered via hyperbaric pressure.

The results of treatment with AS-ICAM-1 and anti-LFA-1 mAb were not additive. There was virtually no difference in wet weight ratio and contraction band necrosis in comparison to AS-ICAM-1 ODN treatment without anti-LFA-1 mAb. There was, however, a decrease in neutrophil infiltration, suggesting that the combination treatment provided a more complete blockade of the specific binding and infiltration of neutrophils mediated by the ICAM-1/LFA-1 interaction. The lack of a synergistic effect could be due to the efficiency of blockade from the hyperbaric administration of AS-ICAM-1 ODN. In addition, the ex vivo application of the AS-ODN to ICAM-1 prior to reperfusion has the added benefit of blocking ICAM-1 protein expression before any is present on the myocardial cell surface, thus giving the potential of a complete blockade of protein expression. Conversely, mAb treatment at the time of reperfusion will only inhibit constitutive proteins expressed near the time of reperfusion.

The advantages of administering AS-ICAM ODN delivery with pressure as a vector are significant. Ex vivo delivery localizes treatment to the organ of interest, thereby limiting systemic effects and reducing the total amount of AS-ODN needed. In addition, because such small amounts of AS-ODN are needed for ex vivo delivery to target organs, there is a much smaller chance of producing a recipient immune reaction than if AS-ODNs or monoclonal antibodies were given systemically [7].

The functional heart model was used in an attempt to provide left ventricular volume loading. The normal heterotopic heart model has the donor aorta anastomosed to the recipient abdominal aorta, and the pulmonary artery anastomosed to the recipient vena cavas. As a result, there is little flow to the recipient left ventricle. The functional heart model shunts the venous blood from the right to left side of the heart, thereby establishing a volume load for the left ventricle to eject against systemic pressure. Consequently, this rodent functional heterotopic heart model may be a better configuration for the assessment of reperfusion injury even though no biochemical differences were observed compared to the standard model. Future studies will use sonomicrometry and left ventricular pressure to evaluate pressure-volume relationships.

In conclusion, ex vivo pressure mediated AS-ICAM delivery either alone or in combination with anti-LFA-1 mAb is a safe and effective method to limit reperfusion injury in cardiac allografts. Additionally, AS-ICAM-1 ODN treatment achieved more of a reduction in reperfusion injury than treatment with anti-LFA-1 mAb, emphasizing the importance of ICAM-1 in ischemia-reperfusion injury. Because of the tissue specificity that is attained and the low amounts of AS-ODN used, pressure mediated gene therapy is a good alternative to the more traditional methods of gene therapy induction such as liposomes and viral vectors. Finally, the early cellular and molecular changes that are achieved with ex vivo blockade of allograft ICAM-1 expression may have long-term beneficial effects such as a decrease in graft coronary artery disease.

	Acknowledgments

 
We would like to thank Timothy Brazelton, BS, and Randall Morris, MD, for their assistance in histologic analysis. Bernard Hausen, MD, was instrumental in modifying the functional heart transplant technique. This work was supported by a grant from the Ralph and Marion Falk Foundation.

	References

Top Abstract Introduction Material and methods Results Comment References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Robert C. Robbins Permission Requests Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Feeley, B. T. Articles by Robbins, R. C. Search for Related Content PubMed PubMed Citation Articles by Feeley, B. T. Articles by Robbins, R. C.