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Circulation Research. 2000;86:166-174

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(Circulation Research. 2000;86:166.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Antisense Intercellular Adhesion Molecule-1 (ICAM-1) Oligodeoxyribonucleotide Delivered During Organ Preservation Inhibits Posttransplant ICAM-1 Expression and Reduces Primary Lung Isograft Failure

Koichi Toda, Koichi Kayano, Ann Karimova, Yoshifumi Naka, Tomoyuki Fujita, Kanji Minamoto, Catherine Y. Wang, David J. Pinsky

From the Columbia University College of Physicians and Surgeons, New York, NY.

Correspondence to David J. Pinsky, Department of Medicine, Columbia University, PH 10-Stem, 630 West 168th St, New York, NY 10032. E-mail djp5{at}columbia.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—Transiently increased expression of leukocyte adhesion receptors after lung preservation contributes to early graft demise by recruiting leukocytes, activating complement, and causing microcirculatory stasis. We hypothesized that inhibiting intercellular adhesion molecule-1 (ICAM-1) expression even briefly may significantly improve lung graft function and that the preservation period might provide a unique window to deliver a therapeutic pulse of antisense oligonucleotide ICAM-1 to inhibit ICAM-1 expression after transplantation. Interleukin-1ß–treated rat pulmonary endothelial cells given a 20-mer phosphorothioate oligonucleotide comprising an antisense span targeted to the 3'-untranslated region of rat ICAM-1 demonstrated an oligonucleotide dose–dependent reduction in ICAM-1 expression. Using a cationic liposomal carrier, this same antisense oligonucleotide (but not the sense control) instilled into the pulmonary vasculature at the time of preservation reduced subsequent graft ICAM-1 expression and graft leukostasis and markedly improved oxygenation, pulmonary blood flow, and graft survival. These experiments demonstrate that the preservation period presents a window during which to target an anti–ICAM-1 expression strategy to inhibit early adhesion receptor expression and improve functional outcome after lung transplantation.


Key Words: intercellular adhesion molecule-1 • lung transplantation • isograft • leukocyte adhesion receptor


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Clinical lung transplantation at its best is a harrowing experience, because lung grafts can fail catastrophically shortly after reperfusion for reasons that are often not understood.1 2 Clinical lung preservation strategies are directed toward maintaining proper electrolyte and osmotic homeostasis, but surprisingly little is done to protect the vast pulmonary vascular network on which the lung depends for both integrity and function. Because the early expression of the leukocyte adhesion receptor P-selectin can result in rapid sequestration of neutrophils (polymorphonuclear leukocytes [PMNs]) after lung transplantation,3 promoting microcirculatory stasis and local tissue destruction, we hypothesized that a strategy that protects the lungs from early leukocyte recruitment could confer clinical benefit. Because P-selectin places neutrophils into a favorable steric relationship for intercellular adhesion molecule-1 (ICAM-1) binding to ß2-integrins, we hypothesized that early inhibition of inducible ICAM-1 expression might serve as a useful target for therapeutic intervention in lung transplantation to prevent acute graft injury. Toward this end, we have focused on the unique opportunity provided by the lung harvest procedure, during which it is possible to deliver agents (such as antisense oligodeoxynucleotides) ex vivo directly into the vasculature, which can block adhesion receptor expression during the first few critical hours after reperfusion. The current studies were designed to (1) elucidate the nature and functional relevance of endothelial ICAM-1 expression in the setting of isogeneic lung transplantation, which has not been established to date, and (2) to test the hypothesis that inhibiting ICAM-1 by pulsed delivery of an effective antisense ICAM-1 oligodeoxynucleotide during lung preservation can effectively block early ICAM-1 expression and improve early lung graft function and survival after transplantation.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Preservation Solution
For all transplant experiments, the basic preservation solution consisted of modified EuroCollins solution, modified by adding magnesium sulfate and glucose. This represents the standard formulation used in clinical lung transplantation.4

Sense and Antisense Oligonucleotides
Sense and antisense phosphorothioate oligonucleotides were chemically synthesized and purified with high-performance liquid chromatography. The ICAM-1 antisense oligonucleotide was designed against the 3'-untranslated region of the rat ICAM-1 gene5 6 and comprised the following sequence: 5'-ACC GGA TAT CAC ACC TTC CT-3'. The complementary sense sequence used was 5'-AGG AAG GTG TGA TAT CCG GT-3'.

Cationic Liposomal Carrier
To transfect the oligonucleotides into cells or pulmonary grafts, a cationic liposomal carrier was used that has demonstrated efficacy and lack of toxicity7 in pulmonary endothelial cells. The liposome formulation chosen for these experiments was the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) to enhance the oligonucleotide uptake by cells.

Endothelial Cell Experiments
Rat pulmonary microvascular endothelial cells were a generous gift of Dr Una Ryan (Avant Immunotherapeutics, Needham, Mass).8 Cells were grown to 60% to 70% confluency in 10-cm Petri dishes and washed before use. The oligonucleotides were prepared for use as detailed in the accompanying electronic supplemental material. The oligonucleotide/lipofectin mixture was added to the cells for 3 hours of incubation, after which recombinant murine interleukin (IL)–1ß was added to the culture medium to a final concentration of 2.5 ng/mL. Cultures were then incubated for an additional 16 hours, at which point cells were harvested for ICAM-1 protein and mRNA measurements.

Lung Transplant Experiments
Experiments were performed according to an institutionally approved protocol and in accordance with AAALAC guidelines.

Donor Lung Harvest
Inbred male Lewis rats were used. Donors were heparinized, after which the right pulmonary artery (PA) was ligated to restrict delivery of preservation solution to the donor lung used for grafting, and 7 mL of 4°C preservation solution was administered into the main PA at a constant infusion pressure of 20 mm Hg. The left lung was then harvested and submerged for 4 hours in 4°C preservation solution.

Transplantation
Orthotopic left lung transplantation was performed as described.9 For all experiments, the preservation duration was identical (4 hours). However, depending on the particular experiment, reperfusion durations ranged from 30 minutes to 24 hours (indicated in the figure legends). For those experiments in which oligonucleotides were studied, the base preservation solution was supplemented with lipofectin followed by the addition of either the sense or the antisense oligonucleotide construct and prepared fresh for each experiment. After lung transplantation, hemodynamic measurements were obtained as previously reported,9 with instrumentation beginning just before the 6-hour reperfusion time point. Thirty minutes after ligation of the native right PA (or at the time of recipient death, if it occurred before 30 minutes), transplanted lung tissue was excised for myeloperoxidase analysis or determining ICAM-1 expression.

Immunoblotting for ICAM-1 Protein
Protein was extracted by washing, scraping, and sonicating cells in the presence of protease inhibitors.10 Integral membrane proteins were extracted using a modification of a previously reported method,11 and protein concentrations determined according to the Bradford method.12 Afterward, samples were prepared for nonreduced SDS-PAGE; ICAM-1 was detected using a primary mouse monoclonal anti-ICAM-1 IgG (1A29 clone), with subsequent densitometric analysis of developed bands.

Northern Blotting for ICAM-1 mRNA
Northern blotting was performed using total cellular RNA, with RNA species of interest detected with either a rat ICAM-1 cDNA probe or a human ß-actin cDNA probe, with subsequent densitometric analysis of developed bands.

Myeloperoxidase Assay
Myeloperoxidase assay was performed using a chromogenic assay, as previously described,13 and data are expressed as change in absorbance at 460 nm.

Immunohistochemistry
Lungs were prepared for immunohistochemistry by submerging the lung tissue in cold saline under pressure to evacuate air and fill alveoli with saline, embedded, frozen, and sectioned. A primary mouse monoclonal anti-rat ICAM-1 IgG was used, after which a secondary fluorescent antibody was applied.

Statistics
Significant differences between groups were tested for with the Mann-Whitney U test. Animal survival data were analyzed by contingency analysis using the {chi}2 statistic. Values are expressed as mean±SEM, with differences considered statistically significant if P<0.05.

An expanded Materials and Methods section is available online at http://www.circresaha.org.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Increased Early Expression of ICAM-1 in the Rat Lung Transplant Model
To determine whether ICAM-1 expression is increased in the setting of lung transplantation, an orthotopic rat lung transplant model9 was used and ICAM-1 expression examined. After transplantation, there was a significant increase in ICAM-1 mRNA levels as early as 30 minutes after reperfusion, apparent both on a representative Northern blot (Figure 1ADown) and on multiple blots (n=5) scanned for relative densitometric calculation (Figure 1BDown). ICAM-1 mRNA levels peaked at 3 hours of reperfusion (171±27% increase versus fresh lung, P<0.01), subsequently tapering off to control levels by 12 hours. Although an insignificant increase in ICAM-1 protein expression was observed at 30 minutes, a significant increase in ICAM-1 protein expression was observed at 3 hours, peaking at 6 hours after reperfusion (179±25% increase versus fresh lung, P<0.01). These data are shown in a representative immunoblot (Figure 1CDown) as well as with a statistical analysis performed on the relative densitometric scans performed on immunoblots from 6 separate experiments (Figure 1DDown). Immunohistochemical examination for ICAM-1 using a primary monoclonal mouse anti-rat ICAM-1 IgG was performed to localize the sites of increased ICAM-1 expression. ICAM-1, which is constitutively expressed at low levels in unperturbed pulmonary endothelial cells and type I alveolar epithelial cells,14 15 was expressed primarily in the structures of the alveolar wall in the fresh lung (Figure 2ADown). However, in the transplanted lung, there was an increased intensity of alveolar wall staining and the appearance of strongly stained microvessels {approx}100 µm in size (Figure 2BDown). When control sections were subjected to immunostaining procedures in the absence of the primary antibody, no staining was observed (data not shown).



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  		Figure 1. A, Effect of reperfusion duration on ICAM-1 mRNA levels in transplanted lungs. Samples were taken from fresh (nonpreserved) lungs or lungs that had been preserved for 4 hours at 4°C, transplanted orthotopically into an isogeneic recipient, and then reperfused for the indicated durations. Total mRNA was obtained and Northern blotting for ICAM-1 performed, with blotting for ß-actin mRNA performed as a control. B, Quantitative time course of relative ICAM-1 mRNA levels after transplantation; data were obtained by densitometric analysis of Northern blots from multiple experiments (n=5 for each time point). Data are expressed as relative increase in ICAM-1 mRNA compared with fresh lung. Values are mean±SEM; *P<0.01. C, Effect of reperfusion duration on ICAM-1 protein expression in transplanted lungs. Samples were obtained from experiments performed as described in panel A and analyzed by immunoblotting for ICAM-1 protein using a primary mouse monoclonal anti–rat ICAM-1 IgG. D, Quantitative time course of relative ICAM-1 protein levels after transplantation; data were obtained by densitometric analysis of Western blots from multiple experiments (n=6 for each time point). Data are expressed as relative increase in ICAM-1 protein compared with fresh lung. Values shown are mean±SEM; *P<0.01.



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  		Figure 2. Immunohistochemical localization of ICAM-1 antigen expression in the lungs. Sites of binding of a primary mouse monoclonal anti-rat ICAM-1 antibody emit an intense green fluorescence when exposed to an excitation wavelength of {approx}475 nm. A, ICAM-1 immunostaining of fresh (nontransplanted) lung. ICAM-1 appears to be expressed primarily in the structures of the alveolar wall. B, ICAM-1 immunostaining of a lung that had been preserved for 4 hours at 4°C, transplanted orthotopically into an isogeneic recipient, and then reperfused for 6 hours. The endothelial lining of small pulmonary vessels stained intensely for ICAM-1, with an increased intensity of alveolar staining as well. Magnification x250.

In Vitro Efficacy of Antisense ICAM-1 Oligonucleotide on Rat Pulmonary Microvascular Endothelial Cells
To test the functional significance of ICAM-1 expression in lung transplantation, and to determine whether the preservation period presents an opportunity for therapeutic administration of an antisense ICAM-1 oligonucleotide, we first tested the in vitro efficacy of a 20-mer phosphorothioate oligonucleotide comprising an antisense span targeted to the 3'-untranslated region of rat ICAM-1.6 As ICAM-1 increases after pulmonary ischemia,16 17 and IL-1ß is a known potent stimulus for endothelial ICAM-1 induction in vitro,14 18 IL-1ß was therefore used as an ICAM-1–inducing stimulus for these studies. After exposure to 2.5 ng/mL of IL-1ß for 16 hours, rat pulmonary microvascular endothelial cells exhibited a strong induction of ICAM-1 mRNA and protein compared with untreated cells (Figure 3Down, leftmost 2 lanes of both panels A and B). Application of the antisense ICAM-1 oligonucleotide in a cationic liposomal carrier (lipofectin) inhibited ICAM-1 protein expression in an oligonucleotide dose–dependent fashion (Figure 3ADown); at the 20 µg/mL dose, antisense ICAM-1 oligonucleotide inhibited the IL-1–induced increase in ICAM-1 expression by 380% (n=5, P<0.05). No significant reduction in ICAM-1 expression was observed in rat pulmonary microvascular endothelial cells treated with IL-1ß and an ICAM-1 sense construct spanning the region identical to the one the antisense construct used (Figure 3ADown, rightmost lane).



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  		Figure 3. Effect of IL-1ß on ICAM-1 expression in rat pulmonary microvascular endothelial cells in vitro and the inhibitory effects of antisense ICAM-1 oligonucleotide. A, Effect on ICAM-1 protein expression, measured by Western blotting using a primary murine anti-rat ICAM-1 IgG. Untreated cells demonstrated some constitutive ICAM-1 protein, which increased after exposure to IL-1ß (2.5 ng/mL) for 16 hours (second lane from left). Using a liposomal carrier, application of antisense targeted to the 3'-untranslated region of rat ICAM-1 demonstrated an antisense-dose–dependent reduction in endothelial ICAM-1 expression. A control experiment in which a sense control was used is shown in the rightmost lane. B, Effect on ICAM-1 mRNA levels. Using the same experimental design as in panel A, separate experiments were performed by Northern blotting for ICAM-1 mRNA using a 2.6-kB cDNA probe for rat ICAM-1 mRNA, with ß-actin used as a control. Treatment with antisense (AS; 20 µg/mL) inhibits the IL-1ß–mediated augmentation of rat pulmonary microvascular endothelial ICAM-1 expression (rightmost lane). Increase in ICAM-1 levels seen in IL-1ß–treated cells (second lane from the left) was unaffected by application of a sense ICAM-1 oligonucleotide (S; 20 µg/mL) but was significantly reduced by antisense (20 µg/mL).

Because antisense ICAM-1 oligonucleotides targeted to the 3'-untranslated region of the ICAM-1 transcript have been shown to decrease mRNA stability and thereby reduce ICAM-1 protein levels,18 19 we examined the effects on mRNA levels of the antisense ICAM-1 oligonucleotide used in the current experiments, which was also targeted to the 3'-untranslated region of rat ICAM-1. These experiments demonstrated that this antisense oligonucleotide reduced the IL-1ß–induced increase in ICAM-1 mRNA by 250% (n=5, P<0.05) (Figure 3BUp). No similar reduction in ICAM-1 levels was observed when the sense construct was used.

Effects of Antisense ICAM-1 Oligonucleotide on Lung Graft ICAM-1 Expression
Using the same antisense ICAM-1 oligonucleotide that had shown in vitro efficacy in rat pulmonary endothelial cells, experiments were performed to determine whether it could inhibit the increased expression of ICAM-1 observed after transplantation. When the control (sense) oligonucleotide in a cationic liposomal carrier was flushed into the pulmonary vasculature at the time of lung harvest and allowed to remain in place for the 4-hour preservation period, ICAM-1 protein expression measured 6 hours after transplantation/reperfusion was markedly increased (Figures 4ADown and 4BDown). The degree of increased ICAM-1 expression in the sense-treated lungs was similar to that seen in untreated lung transplants (Figure 1Up). In sharp contrast, however, when identical procedures were performed using the antisense compound, ICAM-1 protein expression was markedly reduced, down to the background levels seen in untreated/nontransplanted (fresh) lungs (Figures 4ADown and 4BDown). As the in vitro experiments showed that the antisense oligonucleotide reduced ICAM-1 mRNA levels, mRNA levels were also examined in in vivo experiments. These experiments demonstrated that antisense (but not sense) ICAM-1 oligonucleotide blocked the increase ICAM-1 mRNA levels seen after lung transplantation (Figures 4CDown and 4DDown).



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  		Figure 4. Inhibitory effect of antisense ICAM-1 oligonucleotide on pulmonary graft ICAM-1 expression. A, Effect on graft ICAM-1 protein expression, measured by Western blotting using a primary murine anti-rat ICAM-1 IgG. Samples were taken from fresh (nonpreserved) lungs or from lungs that had been treated with 7 mL of 50 µg/mL of either sense (S) or antisense (AS) oligonucleotide (in 50 µg/mL of cationic liposomal carrier) administered directly into the left PA at the time of lung harvest. Pulmonary arterial and venous clamps were placed to prevent fluid extravasation during the 4-hour submersion in 4°C modified EuroCollins preservation solution. Lungs were then transplanted orthotopically into an isogeneic recipient, reperfused for 6 hours, and then excised. Immunoblotting for rat ICAM-1 antigen was performed as described above (a representative blot is shown). B, Quantitative analysis of densitometric scans of multiple immunoblots showing the relative increase in ICAM-1 protein expression (vs fresh) seen when lungs were preserved with sense, but inhibited ICAM-1 expression when lungs were preserved with antisense. n=7 for fresh lungs, and n=12 transplants each for sense and antisense; data are mean±SEM; *P<0.005. C, Effect of sense or antisense on graft ICAM-1 mRNA levels, measured by Northern blotting, with ß-actin as a control. To obtain RNA, separate additional experiments were performed as described in panel A, except that reperfusion duration was 3 hours. Representative Northern blot is shown. D, Relative ICAM-1 mRNA levels were evaluated in multiple blots by densitometry and expressed as relative increase compared with fresh (nonpreserved) lung samples. n=2 for fresh lungs and n=4 transplants each for sense and antisense groups; data are mean±SEM; *P<0.05.

Effects of Preservation-Delivered Antisense ICAM-1 Oligonucleotide on Posttransplantation Neutrophil Accumulation and Graft Function
To gauge the functional effects of ICAM-1 expression and its blockade by antisense ICAM-1 oligonucleotide on pulmonary graft leukosequestration, myeloperoxidase activity was determined in (1) freshly harvested and transplanted isografts, as well as isografts subjected to hypothermic preservation after treatment with either (2) the cationic liposomal carrier to which no oligonucleotides were added or to which either (3) sense or (4) antisense ICAM-1 oligodeoxyribonucleotides were added. These experiments demonstrated a significant reduction in graft neutrophil accumulation by the antisense-treated, but not the sense-treated, grafts, suggesting that the antisense compound inhibited the expression of functional ICAM-1 protein (Figure 5ADown). To establish that the inhibition of ICAM-1 expression by the antisense compound is pathophysiologically significant in terms of protecting the graft against primary failure, experiments were performed using the rat lung transplant model in which, after transplantation, the nontransplanted lung was removed from the pulmonary circuit by ligating the right PA (as previously described).3 9 20 21 22 23 24 This ensures that subsequent functional measurements (including recipient survival) are entirely dependent on the function of the transplanted lung. In these experiments, treatment of the lung graft at the time of harvest with antisense ICAM-1 oligonucleotide resulted in significant improvements in arterial oxygenation (Figure 5BDown), reductions in pulmonary vascular resistance (Figure 5CDown) with increased pulmonary blood flow (5.5±2.8 versus 11.0±2.9 mL/min for sense versus antisense, respectively, P<0.05), and markedly improved recipient survival (Figure 5DDown). In sharp contrast, treating lungs with either the liposomal carrier alone or the carrier to which the sense compound was added did not inhibit ICAM-1 expression; grafts so treated were associated with poor functional and survival outcomes after lung transplantation.



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  		Figure 5. In vivo functional effects of antisense ICAM-1 oligonucleotide in lung transplantation. Lungs were harvested and transplanted immediately (fresh, n=6, comprising untreated/nonpreserved isografts) or subjected to hypothermic preservation after treatment with vehicle+cationic liposomal carrier alone (Control, n=6; prepared as described in Materials and Methods) or vehicle to which either sense (S, n=12) or antisense (AS, n=12) ICAM-1 oligonucleotides were added. At 6 hours after reperfusion, the native right PA was ligated to allow examination of the function of the preserved/transplanted left lung independently of the native right lung. After the ligation procedure, hemodynamics were recorded until the final time at which the recipient was alive, or at the 30-minute time point if the animal survived until then, at which point the animal was euthanized and lung tissue collected. A, Effects on pulmonary graft neutrophil sequestration, measured with an assay of myeloperoxidase activity (MPO; {Delta}Abs 460 nm/min). B, Effects on pulmonary graft gas exchange, measured as arterial oxygenation (PO2, mm Hg); recipients were ventilated with 100% O2 throughout the posttransplant period). C, Effects on pulmonary graft vascular resistance (mm Hg/mL min–1), measured by recording pulmonary arterial blood flow, pulmonary arterial pressure, and left atrial pressure. D, Effects on recipient survival; data are mean±SEM; *P<0.05.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Primary lung graft failure, which occurs in up to 20% of all cases shortly after transplantation, is a catastrophic clinical event that occurs unpredictably and for which there is no effective treatment.1 2 25 Because as an organ, the lung is one of the most richly vascularized, with nearly 40% of its parenchymal cellular mass composed of endothelial cells,26 it is not surprising that endothelial activation during the preservation or early transplantation period may represent a significant cause of primary lung graft failure. Because the lungs are detached from their native blood supply during the mechanical lung harvest procedure, we hypothesized that this would present an opportune time to deliver a therapeutic agent directly to endothelial cells to prevent their activation during or shortly after preservation. As we have shown neutrophil adhesion to activated endothelium is a culprit in early graft demise,3 27 28 we hypothesized that ICAM-1 would be a suitable target for genetic manipulation of the graft vasculature, because its expression is inducible. However, in the setting of primary lung graft failure after lung transplantation, it had not been previously established that ICAM-1 expression is augmented or relevant. In the current experiments, we demonstrate that ICAM-1 expression increases within the first several hours after lung transplantation and that this increase is pathophysiologically important. Furthermore, by administering an anti–ICAM-1 antisense oligonucleotide directly into the pulmonary vasculature at the time of harvest, we can virtually obliterate the posttransplant increase in ICAM-1 expression and effect a remarkable improvement in graft function and recipient survival compared with sense-treated controls. These data indicate that there exists a unique opportunity for genetic manipulation of the explanted graft vasculature that can translate into real, functionally relevant effects.

In the experiments performed in this study, an antisense approach was taken to reduce lung graft ICAM-1 expression. In this approach, hybridization of an antisense oligonucleotide to the complementary sense strand by normal Watson-Crick base pairing can block protein synthesis by steric hindrance causing translational arrest, inhibit RNA processing, and/or alter the susceptibility of targeted mRNA to degradation.29 The choice of using the 3'-untranslated region for the antisense oligonucleotide in our experiments was based on several published reports, in which an antisense oligonucleotide targeted to this region of human ICAM-1 was particularly effective at specifically inhibiting ICAM-1 expression,18 19 as well as a report showing efficacy in a renal ischemic injury model in the rat.6 The phosphorothioate composition of the oligonucleotide was selected to increase the stability of the compound against serum and other nucleases.30 31 To optimize conditions for study in the rat lung transplant model, we first used in vitro conditions with stimulated rat pulmonary microvascular endothelial cells. In this development stage of our project, IL-1ß was chosen as the ICAM-1–inducing stimulus because it is a known potent inducer of endothelial ICAM-114 18 and because it may be relevant in the setting of pulmonary ischemia and reperfusion injury.32

The cationic liposomal carrier delivery method we chose to use was selected because of the theoretical concerns that viral delivery systems, even though effective in delivering cytokines to the lungs,33 could promote postischemic vascular inflammation,34 35 which we wished to suppress, and because of reports that relatively high levels of transfection efficiency might be possible with the DNA/liposomal method.36 In addition, DNA/cationic liposomal complexes have been shown to successfully deliver genes to the lungs after aerosol or intravenous delivery without adverse effects in terms of pulmonary histology, lung compliance, or alveolar-arterial oxygen gradient.7 In mice, intravenous delivery of a reporter gene as DNA-liposome complexes was far more efficient than use of plasmid DNA alone.36 In a recent study,37 in which the transfection efficiency of a transforming growth factor-ß (TGF-ß) cDNA was examined in a rat lung transplant model, the use of the cationic liposomal carrier was shown to increase transfection efficiency up to 4-fold compared with noncomplexed TGF-ß cDNA. These experiments differ from those in the current study in that a larger cDNA was used, rather than a shorter oligodeoxynucleotide, and the goal of the latter experiments was to increase expression of the administered transgene (TGF-ß). In the experiments shown here, a small oligodeoxyribonucleotide was given, with the intent of inhibiting expression of an endogenous protein whose expression increases markedly after lung ischemia-reperfusion injury, with deleterious consequences. The ex vivo method of delivery was chosen because phosphorothioate oligonucleotides have been shown to activate complement (in a non–sequence-specific manner) when given intravenously.38 We wished to avoid complement activation, as we have previously shown in the same rat lung transplant model that this compounds postischemic lung injury.24

The antisense ICAM-1 oligonucleotide we used in the current experiments not only reduced ICAM-1 protein levels, but ICAM-1 mRNA levels as well. In a recent study in which the molecular mechanisms by which antisense oligonucleotides inhibit ICAM-1 expression were examined, it was shown that human ICAM-1 antisense oligonucleotides inhibited ICAM-1 expression through 2 distinct posttranscriptional mechanisms. In the first, antisense oligonucleotides corresponding to the AUG translation initiation codon mask the ribosome recognition site and prevent the formation of the translation complex. In the second mechanism, an antisense oligonucleotide corresponding to the 3'-untranslated region of ICAM-1 mRNA (targeting similar to that of the antisense construct used in the current experiments) caused a marked increased susceptibility of ICAM-1 RNA to hydrolysis by RNase H, a ubiquitous enzyme that degrades the RNA strand of RNA-DNA hybrid molecules.18 Although experiments using antisense oligonucleotides must be interpreted carefully because of potential non–sequence-specific effects, these are not likely to apply to the current experiments for several reasons. First, we observed a direct inhibitory effect of the antisense oligonucleotide on both mRNA and protein levels of the targeted sequence (ICAM-1), which was not seen with control (sense) oligonucleotide. Although non–sequence-specific effects have been reported on the basis of the binding of phosphorothioate oligonucleotides to growth factors39 40 41 or to elements of the extracellular matrix,42 the lack of 4 consecutive guanosine residues (G-quartet) in the sequence used in the current experiments reduces the possibility of an aptameric effect.

In addition to demonstrating in vivo efficacy of an antisense ICAM-1 strategy in a clinically relevant scenario (lung transplantation), the experiments in this study offer the first direct proof of a functionally deleterious role for ICAM-1 expression in the setting of primary lung graft failure. Although selectins are responsible for initially decelerating circulating PMNs during their transit through the pulmonary vasculature, bonds formed between selectin glycoproteins on endothelial cells and their oligosaccharide counterligands on PMNs form and break readily to permit the PMN to roll to a stop along the endothelial surface. Selectins therefore position the PMN and the endothelial cell into a correct steric relationship so that the more potent adhesion receptor, ICAM-1, can interact with its PMN counterligands, LFA-1 and Mac-1. In fact, static incubation of activated PMNs on artificial lipid bilayers enables comparison of ICAM-1 and P-selectin–dependent adhesive forces; PMN binding through the integrins LFA and Mac-1 to ICAM-1 is 100-fold more shear resistant than binding via CD62 (P-selectin).43 For these reasons, although selectin blockade can also be effective to diminish pulmonary graft leukostasis,3 blockade of ICAM-1 expression appeared to be a particularly attractive target to improve lung preservation. In addition, the pulmonary reperfusion microenvironment exhibits transient and inhomogenous alterations in shearing forces that can transiently upregulate ICAM-1 expression.44 A rat model was chosen for study, because although adhesion receptor null mice have proven extremely useful for studying the pathophysiological roles of various adhesion receptors,45 lung transplantation is not technically possible in the mouse.

Although the present studies do not permit a direct comparison of the effects of functional blockade of P-selectin–dependent adhesion reactions via an antibody approach and the effects of an anti–ICAM-1 expression strategy provided by the antisense ICAM-1 approach, some rough comparisons can be made. In our previous work,3 in which we found that an antibody to P-selectin was effective at reducing the consequences of pulmonary ischemia/reperfusion injury, we looked at a 30-minute time point in the rat model (as P-selectin is rapidly expressed). However, in the current work, data show that ICAM-1 expression does not peak until 3 to 6 hours of reperfusion have passed, and we therefore examined the functional effects of ICAM-1 inhibition at the delayed (6-hour) time point. In Figure 5DUp, our data show that inhibiting ICAM-1 expression does not make a functional difference at the early (30-minute) time point, as would be expected from the time course of expression that we demonstrate in Figure 1Up. With P-selectin blockade alone and an experimental n of 4 (using the same model, but with a longer observation period), the mean survival time is 6 hours; with antisense ICAM-1, 75% of animals survive to 6 hours and presumably beyond (experiments were terminated according to protocol at 6 hours), suggesting that the antisense ICAM-1 approach may be more effective than that of providing a functionally blocking anti–P-selectin antibody.

There are other compelling reasons why we chose to study ICAM-1 in the setting of lung transplantation. Clinical studies have shown increased levels of soluble ICAM-1 after cardiopulmonary bypass, which causes pulmonary circulatory stasis and thereby exposes the lung to ischemia,46 as well as increased expression of ICAM-1 in posttransplant lung biopsy specimens.47 In other transplantation scenarios, such as cardiac28 48 or liver,49 ICAM-1 expression has also been implicated as a pathophysiological mediator of acute graft injury. In an in situ model of lung ischemia and reperfusion, ICAM-1 was shown to be upregulated within the pulmonary microvascular circulation and to contribute significantly to lung reperfusion injury.16 17 In the kidney, ICAM-1 expression is thought to be a mediator of postischemic renal injury and failure. In fact, a recent study showed that systemic (intravenous) administration of an antisense ICAM-1 oligodeoxynucleotide attenuates renal reperfusion injury and renal failure.6 The current experiments, however, differ from the work of Haller et al6 in that not only were different models and modes of antisense oligonucleotide administration used (ex vivo under hypothermic preservation conditions in the current study), but an entirely different vascular bed was the subject for study. The lungs are far more vulnerable to ischemia-reperfusion injury than are the kidneys; for instance, renal preservation may be successful after 24 hours, but lung grafts may not be used after 4 to 6 hours of hypothermic preservation because of the high incidence of primary lung graft failure. In addition, there can be significant differences between the phenotype of endothelial cells between vascular beds. For instance, the stimulus-specificity and selectin-dependence of leukocyte/endothelial adhesion differs between the pulmonary and the systemic vascular beds; in a model of muscle ischemia/reperfusion injury, local injury to the muscle was shown to be selectin-independent, yet injury to the lungs was selectin-dependent.50 In the case of integrins, the CD11/CD18 complex mediates PMN adherence in the systemic circulation, but in the pulmonary circulation, depending on the inciting stimulus, neutrophil adherence may be CD18-independent.51 Although our group has shown using ICAM-1 null mice that cardiac ischemia-reperfusion injury is ICAM-1–dependent,28 others have shown in a lung injury model (using control and ICAM-1–deficient mice) that cobra-venom factor–induced lung injury is ICAM-1–independent.52 A recent review by Rosenberg and Aird53 also provides insights into how the coagulant phenotype of the vessel wall can differ between vascular beds.

There are several theoretical reasons why an antisense approach to inhibit ICAM-1 expression may be preferable to an antibody-mediated approach. Because functionally blocking anti-ICAM-1 antibodies compete with integrin epitopes for binding, it may be difficult to achieve high enough antibody levels in the local ischemic vascular milieu (because of vasoconstriction, thrombosis, and cellular obstruction of microvascular lumina) to fully inhibit endothelial-neutrophil interactions. In addition, binding affinity and kinetics differ between various antibodies, which may account for experimental variability between them in different studies. In an isograft lung transplant model in which functionally blocking anti–ICAM-1 and anti–ß2-integrin antibodies were studied, individual antibodies directed solely against ICAM-1, CD11a, or CD18 were far less effective than a combination of antibodies against all 3.54 In other studies, although an anti–ICAM-1 antibody approach did seem to improve the ability of isolated-perfused rabbit lungs to oxygenate, pulmonary vascular resistance, airway resistance, and lung edema were not improved, suggesting only partial effectiveness of the anti–ICAM-1 approach tested.55 Similar incomplete improvement of lung graft function was seen in a canine single-lung transplant model in which an antibody to CD18 was studied.56 Other potential detractions of an antibody-mediated approach include the potential for Fc-mediated activation of complement (as has been shown in liver transplantation).49 It is also possible that nonlocalized inhibition of ICAM-1–mediated leukocyte-endothelial interactions with an antibody may complicate issues related to clinical infection and immunosuppression.57 58

The experiments shown here are the first to demonstrate a role for antisense ICAM-1 delivered during the preservation period to mitigate acute lung graft injury in the absence of an allogeneic immune response. Whether or not this approach will be useful in lung allograft transplantation to reduce either primary graft failure or rejection remains an open question at this time. Although this is an extrapolation, it is interesting to speculate that the early suppression of graft ICAM-1 expression may have a longer-term immunomodulatory role. Blocking ICAM-1 expression with an antisense oligodeoxynucleotide (given intravenously over 1 to 2 weeks) in the setting of cardiac allograft transplantation was shown to have more of an immunomodulatory effect (to reduce rejection) rather than to reduce immediate graft injury and primary graft failure.59 60 61 In other experiments, anti–interferon-{gamma} antisense oligodeoxynucleotides indirectly reduced ICAM-1 expression as well as cell surface induction of major histocompatibility complex (MHC) class I and II molecules.62 63 However, this alternative mechanism of action (interferon-{gamma} inhibition) is not likely to apply to the current model, in which isogeneic grafts (seen as "self" by the recipient) were used, diminishing a potential intermediary role for MHC class I or II molecule inhibition in reducing acute graft injury. Although in these experiments, the activity and potency of the oligonucleotide to inhibit induction of MHC class I and II molecules was sequence-dependent, the oligodeoxynucleotide appeared to exert its effects by inhibiting the association of interferon-{gamma} with the cell surface. In the current studies, identification of diminished target (ICAM-1) mRNA and protein expression mitigates the need to invoke such alternative mechanisms of action for the ICAM-1 antisense oligodeoxynucleotide.

In summary, the data presented in this paper define the time course of ICAM-1 expression after lung transplantation, which is related to allogeneic-independent factors, and show for the first time the functional relevance of ICAM-1 expression in primary lung graft failure. Furthermore, guided by in vitro pulmonary endothelial cell experiments, this work identifies the pulmonary preservation period as a unique window of opportunity during which to deliver an antisense oligonucleotide to inhibit pulmonary ICAM-1 expression and to improve functional outcome after lung transplantation.


*    Acknowledgments
 
This work was supported in part by the United States Public Health Service (Grants R01 HL55397 and R01 HL60900). We thank Hui Liao and Shoichi Maruyama for their expert suggestions during the course of this work.

Received September 2, 1999; accepted October 27, 1999.


*    References
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