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Ann Thorac Surg 1997;64:1004-1012
© 1997 The Society of Thoracic Surgeons
Departments of Cardiothoracic Surgery and Pathology, Stanford University School of Medicine, Stanford, California
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Abstract |
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Methods. Hearts were harvested from donor rats before and after pretreatment with lipopolysaccharide at -24 hours, underwent 45 minutes of cold ischemia, and were transplanted into ACI recipients with or without anti–ICAM-1 monoclonal antibody treatment. Grafts were procured early for analysis of ICAM-1 expression and reperfusion injury or the recipients were treated with cyclosporin A (to allow long-term graft acceptance) for postoperative days 0 through 9 with procurement on postoperative day 90 to histologically score for chronic graft vascular disease.
Results. Lipopolysaccharide-pretreated PVG heart grafts showed increased ICAM-1 expression by Northern blot and immunohistochemical analysis leading to increased reperfusion injury as assessed by neutrophil infiltration (myeloperoxidase), cardiac edema (percentage wet weight), and histologic injury (percentage area of contraction band necrosis), which was reversed by recipient treatment with anti–ICAM-1 monoclonal antibody. After administration of cyclosporin A, 5 mg/kg for 10 days, lipopolysaccharide-treated grafts had significantly worse chronic graft vascular disease scores (2.56 ± 0.57 versus 1.84 ± 0.75; p < 0.05 by Mann-Whitney U test).
Conclusions. The induction donor inflammatory state before harvest leading to increased cardiac ICAM-1 expression promotes reperfusion injury and chronic graft vascular disease after transplantation in this rodent heterotopic heart model.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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In addition to having little effect on CGVD, currently used immunosuppressants also do not affect primary cardiac graft dysfunction secondary to global ischemia and reperfusion injury (RI). Standard methods of myocardial preservation have generally limited the direct morbidity from primary cardiac dysfunction after relatively short ischemic periods [4]. Nonetheless, there are two reasons to remain concerned about this process. Given the shortage of organs available for clinical transplantation, hearts are often exposed to cold ischemic arrest periods of greater than 3 hours during the organ procurement process, increasing the risk of significant RI after implantation [5]. More importantly, there is accumulating experimental and clinical evidence of a cytokine–adhesion molecule cascade in postischemically reperfused allografts that augments intragraft allorecognition and alloactivation, therefore promoting allograft rejection. This hypothesis is supported by the clinical observation of higher rates of acute [6] and chronic rejection [7] in grafts experiencing worse RI.
Organs for clinical transplantation are often procured from donors who have recently suffered severe trauma or brain death, conditions that have been shown to increase the systemic release of inflammatory cytokines [8]. These cytokines, which are known to upregulate the expression of cell-surface adhesion molecules such as intercellular adhesion molecule (ICAM-1) in vitro [9, 10], have been hypothesized to promote similar changes in donor grafts in vivo [3, 11]. Reperfusion injury that occurs after global cardiac ischemia is thought to be mediated in major part by the accumulation of activated neutrophils, leading to the release of toxic intermediates such as oxygen free radicals, proteases, and activated complement. Upregulation of cell-surface molecules on the graft such as ICAM-1 not only leads to the recruitment of these cells [12], but has been shown to be necessary for neutrophil-mediated cellular damage [10]. The resulting microenvironment at the neutrophil–target cell interface promoted by the interaction of ICAM-1 with its ligands, MAC-1 (CD11b/CD18) and leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18), is likely required for the toxic intermediates to fully exert their damaging effects.
Our previous studies using the PVG to ACI rodent heterotopic heart transplantation model have demonstrated that induction of a systemic inflammatory state led to increased ICAM-1 expression in donor hearts before transplantation. Additionally, there was an earlier and more intense upregulation of ICAM-1 after transplantation during reperfusion compared with hearts procured from healthy donors [11]. This increased ICAM-1 expression was found to predispose hearts to worse reperfusion injury. Other studies in the PVG to ACI strain combination have shown that normal PVG hearts develop increasing rates of CGVD in the setting of decreasing inflammation at 90 days after an initial short course of cyclosporin A (CSA) to these "low-responder" ACI recipients [13]. The current study was designed to test the hypothesis that increased reperfusion injury leads to higher rates of CGVD in the PVG to ACI model.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Animals
Adult male (8 to 10 weeks old, 230 to 270 g) PVG (RT1u) and ACI (RT1a) rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). All animals were maintained in the animal care facilities of the Department of Cardiothoracic Surgery (Stanford University Medical Center, Stanford, CA). Their environment was maintained at 21° ± 2°C with a time-regulated light period from 7:00 AM to 7:00 PM. Rats were provided water and dry food ad libitum. Periodic serologic analysis of room sentinel animals showed that all rats were free of acute viral infection. 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 National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985).
Procedures
PVG donor rats that were untreated (n = 45) or pretreated (n = 57) with LPS (10 mg/kg intraperitoneally) 24 hours before cardiac procurement (-24 hours) were anesthetized on the day of operation (time 0) with methoxyflurane (inhalational) followed by sodium pentobarbital (50 mg/kg intraperitoneally). Donor hearts were harvested after cardiac arrest by coronary perfusion with Stanford Cardioplegia by infusion proximal to an aortic cross-clamp. After explanation, heart grafts underwent a period of cold ischemia in saline solution at 4°C for 45 minutes before heterotopic grafting into the abdomens of ACI recipients that were either untreated or pretreated with 1A29 mAb 2 mg/kg intravenously given just before cross-clamping of abdominal vessels. Total ischemic times including the period required for surgical implantation ranged from 58 to 67 minutes. Heterotopic hearts were then either harvested at 6, 12, and 24 hours after transplantation to assess reperfusion injury (acute recipient) or the recipients were treated with CSA for 10 days to avoid acute rejection and allow for the development of CGVD (chronic recipient). The cardiac allografts were then explanted at 90 days for assessment of CGVD. For RI studies, hearts were immediately flushed after explanation with 2 mL cold phosphate-buffered saline solution at 0.5 mL/min before analysis of ICAM-1 expression by both Northern blot and immunohistochemical techniques and analysis of RI using cardiac edema, neutrophil infiltration, and histologic parameters.
Northern Blot
After perfusion with ice-cold phosphate-buffered saline solution ex situ, native ACI and heterotopic PVG hearts were cleaned of surrounding tissue, snap frozen in liquid nitrogen, and then homogenized in guanidium isothiocyanate followed by pelleting through cesium chloride. Total RNA (30 mg) was then electrophoresed through 1.2% agarose/formaldehyde gels and blotted onto nylon membrane overnight by capillary transfer. Complementary DNA probes for mouse ICAM-1 (ATCC, Rockville, MD) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Clontech, Palo Alto, CA) were radiolabeled with [32P]dCTP by the random-primer labeling method. After ultraviolet cross-linking, RNA immobilized on the membrane was hybridized with the cDNA probes at 42°C for 6 hours. The blots were then washed under increasingly stringent conditions using 1 x SSC, 0.1% sodium dodecyl sulfate at room temperature followed by 0.5 x SSC, 0.1% sodium dodecyl sulfate at 42°C for 15 minutes. Autoradiography was then performed by exposing the blots to Kodak X-OMAT film with Dupont (Wilmington, DE) Cronex Lightening Plus intensifying screens at -80°C for 1 to 5 days.
Immunohistochemistry
Procured and flushed hearts were immediately snap frozen in liquid N2 in OCT embedding compound (Miles, Elkhart, IN) and stored at -80°C. After bringing the sample to -20°C, 6-µm thin sections were placed onto poly-L-lysine–precoated slides (Sigma Diagnostics, St. Louis, MO). Intercellular adhesion molecule-1 was stained using the avidin-biotin-complex method outlined in the Histostain SP immunohistochemistry kit (Zymed Laboratories, South San Francisco, CA). Briefly, sections were air dried at room temperature and fixed in acetone at -20°C for 10 minutes. Sections were rehydrated in 1% bovine albumin–phosphate-buffered saline solution for 10 minutes, then incubated with the primary antibody 1A29 (gift of M. Miyasaka, Osaka, Japan) for 45 minutes followed by a biotinylated goat anti-mouse immunoglobulin G (Zymed) for 15 minutes. The avidin-biotin complex was applied and diaminobenzidine tetrahydrochloride was used as the chromogen. The substitution of 1% bovine albumin–phosphate-buffered saline solution for the primary antibody served as the negative (reagent) control. Rat cervical lymph nodes and cardiac allografts at day 3 in untreated recipients served as the positive control. Sections were scored for ICAM-1 staining intensity by a pathologist blinded as to experimental group.
Myeloperoxidase
Cardiac tissue was prepared for analysis of myeloperoxidase (MPO) levels by being homogenized on ice in 2 mL of 0.5% hexadecyltrimethyl ammonium bromide (Sigma) in 50 mmol/L KPO4 (Sigma) at pH 7.0, followed by sonication for 10 seconds, three freeze/thaw cycles, and resonication. Final supernatant for analysis was obtained by centrifugation at 12,000 g for 15 minutes. The supernatant (0.035 mL) was transferred into 3 mL of reagent buffer consisting of 50 mmol/L KPO4, 100 mmol/L guaiacol (Sigma), and 0.0017% H2O2 (Sigma), adjusted to pH 7.0 at 25°C with 1 mol/L KOH. At 1 minute, the change in absorbance at 470 nm wavelength (
A470nm) was read using a DU-50 Spectrophotometer (Beckman Instruments Inc, Irvine, CA). Varying concentrations of purified MPO enzyme (Sigma, supplied at 10 U/mL) were used to provide a standard curve. The amount of MPO per milliliter of sample was calculated by dividing the A470nm after 1 minute by the amount (0.035 mL) and protein concentration (determined using a BCA protein assay kit, Pierce Pharmaceuticals, Rockford, IL) of the sample used.
Percentage Wet Weight
The atrial caps were removed and then grafts were divided into halves along the long axis. After removal of intraventricular clot, hearts were weighed before and after drying in an oven at 100°C for 24 hours. Percentage wet weight (% wt/wt) was calculated according to the following equation: % wt/wt = (wet weight - dry weight)/wet weight x 100.
Percentage Area of Contraction Band Necrosis
Procured grafts were sectioned perpendicular to the long axis of the heart and fixed in buffered formalin for 24 hours. Trichrome-stained sections taken from paraffin-embedded samples were reviewed with a pathologist and then assessed for total area involved in contraction band necrosis using a computer-assisted image analysis system (C-imaging Systems, Cranberry Township, NJ).
Evaluation of CGVD
Grafts were removed for histologic analysis of CGVD on POD 90. After harvest, grafts were sectioned perpendicular to the long axis of the heart and fixed in buffered formalin for 24 hours. Thin hematoxylin and eosin and elastic van Gieson stained sections of paraffin-embedded samples were examined by a pathologist (M.B.) blinded as to experimental group and were assigned a CGVD score. This score was the mean score for all the vessels in a section and therefore represented the fact that normal and occluded vessels were often found in the same sections (ie, displayed high standard deviations). Individual vessels were subjected to a five-point grading scale from 0 to 4 (0 for no involvement, 1 for partial intimal involvement, 2 for concentric intimal thickening, 3 for more severe concentric involvement up to 50% luminal narrowing, and 4 for greater than 50% up to complete occlusion).
Statistical Analysis
The parameters of RI—neutrophil infiltration (MPO), cardiac edema (%wt/wt), and histologic injury (%CBN)—were compared at their respective time points (6, 12, or 24 hours) by means of the two-tailed Student's t test. Chronic graft vascular disease scores between the four groups (grafts from donors with or without LPS pretreatment and in recipients treated with CSA 5 or 10 mg • kg-1 • day-1 x 10 days) were compared using unpaired analysis of variance with Dunnett's post test. The incidence of acute rejection of PVG grafts between groups was compared using Fischer's exact test. Significance was assigned to p values less than 0.05. Data are expressed as mean ± standard deviation.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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. At the mRNA level, Northern blots (Fig 1) of native PVG hearts harvested 6 hours after LPS injection showed strong 2.8-kb ICAM-1 messenger RNA bands. These homogenates served as the positive control. Hearts harvested from healthy PVG donor rats failed to reveal the 2.8-kb messenger RNA band as shown in the positive control lane at all postoperative time points. However, hearts harvested from LPS-treated donors demonstrated weak ICAM-1 bands after 12 and 24 hours of reperfusion. At these time points, ICAM-1 bands in the lanes from grafts are darker than those seen in the lanes from native nontransplanted hearts, which serve as the internal negative control. At the protein level, nontransplanted hearts from healthy donors showed weak ICAM-1 staining on the capillary and venous endothelium (Fig 2) by immunohistochemical analysis. This pattern did not change after reperfusion of these hearts for 6 or 12 hours. However, after 24 hours of reperfusion weak ICAM staining was seen on the intercalated disks of myocytes. Graft harvested from LPS-pretreated donors before transplantation showed ICAM-1 staining on the endothelium of arteries and more intense staining on the veins. As in the healthy heart, no ICAM-1 staining was seen on myocardial cells before transplantation. However, this lack of myocardial staining was in contrast to an intense upregulation in myocardial staining in these LPS-pretreated grafts after 24 hours of reperfusion.
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). Statistical significance was reached at the 12-hour time point using Student's t test (0.33 ± 0.24 versus 0.05 ± 0.04, n = 5 each group). Cardiac edema was also increased at all time points in hearts from LPS-treated donors compared with healthy donors with statistical significance again achieved at 12 hours (81.67 ± 1.86 versus 79.24 ± 0.71, n = 5 each group) (Fig 3B). A computer-assisted image analysis system was used to quantitate the amount of contraction band necrosis in histologic sections taken from hearts harvested from LPS-treated donors compared with hearts from healthy donors. At 12 and 24 hours of reperfusion, this area was greater in grafts from LPS donors with statistical significance reached at 24 hours (15.23 ± 1.89 versus 11.35 ± 2.03, n = 5 each group) (Fig 3C). All three parameters of injury were significantly reduced in grafts procured from LPS-pretreated donors after a single perioperative injection of 1A29 mAb (2 mg/kg intravenously) to the recipient (0.07 ± 0.03 versus 0.33 ± 0.24 for MPO, n = 5; 78.98 ± 0.93 versus 81.67 ± 1.86 for %wt/wt, n = 5; 12.03 ± 1.52 versus 15.23 ± 1.89 for %CBN, n = 5).
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). Lipopolysaccharide-pretreatment of grafts had no effect on CGVD scores in recipients treated with higher dose (10 mg • kg-1 • day-1 x 10 days) CSA (1.35 ± 0.82, n = 5 for LPS-pretreated grafts versus 1.35 ± 0.73, n = 9 for healthy grafts).
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Both clinical and experimental evidence provide support for the idea that an initial nonspecific injury mediated by ischemia/reperfusion contributes to the eventual development of chronic graft failure secondary to CGVD. Certain antioxidant substances such as superoxide dismutase (SOD) have been shown in animal models to improve early graft dysfunction secondary to RI after transplantation [16, 17]. In a prospective, randomized double-blind trial in renal transplant patients, recombinant human SOD was found to exert a beneficial effect in terms of chronic rejection events and long-term graft survival [18]. Given that SOD does not have direct immunosuppressant properties, the presumed mechanism of this effect was by interruption of the cytokine–adhesion molecule cascade. By preventing accumulation of reactive oxygen intermediates produced by infiltrating neutrophils, recombinant human-SOD likely prevented endothelial activation with upregulation of adhesion molecules and further neutrophil infiltration. Further subgroup analysis showed that the effectiveness of rh-SOD treatment on improving long-term graft survival was primarily with those individuals who experienced acute rejection episodes. Taken in combination with the increased CGVD seen in our study only in the group of rats with higher rates of both acute rejection and RI (LPS-pretreated donor and 5 mg • kg-1 • day-1 CSA), these data emphasize the accelerating effect that these processes have together on chronic transplant failure. The improved long-term survival results of renal grafts procured from living nonrelated donors compared with cadaveric donors [19], together with the increased rates of acute [6] and chronic [7] rejection seen in human transplant recipients experiencing an initial severe bout of RI, provide further clinical support for this idea.
Increased donor graft ischemic times in rodent heterotopic heart [20] and aortic allograft [21] models have been shown to promote the development of vascular narrowing similar histologically to that seen in human CGVD. However, ischemia itself can cause direct vascular injury [22], thereby complicating the issue of whether ischemic injury or the resulting increased postischemic RI is more important in the development of CGVD. Our studies were designed so that ischemic times were held constant between groups with increased neutrophil-mediated RI documented only in the LPS-pretreated group. This allows a specific correlation of the severity of RI with CGVD independent of any additional confounding effects of ischemia.
In addition, our model implicates ICAM-1 as playing a specific role in the subsequent development of CGVD. There is both direct and indirect corroborating evidence that the level of expression of ICAM-1 in allografts influences chronic rejection. Using the Lewis to Fischer (F344) renal transplant model, Hancock and colleagues [23] correlated a change in pattern and increase in ICAM-1 staining intensity using immunohistochemistry with the histologic onset of chronic rejection changes at 12 weeks in Lewis allografts. In human heart allografts, similar correlations between a late onset of ICAM-1 upregulation and early signs of CGVD have been seen in both clinical [24] and autopsy [25] studies. The key role of ICAM-1 in the RI noted in various animal models and human transplant recipients provides further indirect evidence of involvement in CGVD. Reperfusion injury leads not only to primary allograft dysfunction, but also to the induction of class I and II major histocompatibility complex [26] and other adhesion molecules, which is thought to lead to first passage sensitization of alloreactive T cells and induction of both acute and chronic rejection activity [27].
The conclusions that can be drawn from this study regarding the role of ICAM-1 upregulation in RI and CGVD are restricted by certain limitations. First, neutrophil infiltration, cardiac edema, and histologic injury provide only indirect evidence of RI. Assessment of left ventricular function, specifically, the maximum rate of increase of left ventricular pressure, by placement of an intraventricular balloon before reperfusion has been used in other studies [28] to provide a more direct demonstration of the clinical relevance this heterotopic heart model. These studies are ongoing in our laboratory. Second, an alternative explanation for the differences in CGVD scores between LPS-pretreated and healthy donor grafts seen only in the low-dose CSA group could be that the absorption after gavage administration was erratic, leading to significant differences in immunosuppression. Although we did not monitor and therefore cannot rule out differences in CSA levels between the two low-dose groups, we believe that increased RI played the more important role in promoting RI for the following reasons: (1) acute rejection was not different between the two groups receiving low-dose CSA (33% for LPS-pretreated versus 29% for healthy grafts), and (2) the sample sizes were relatively large (n = 18 and 17), which likely would have controlled for erratic absorption between groups. Finally, we chose to specifically investigate the effects of LPS donor treatment on ICAM-1 expression. However, this treatment has been documented to upregulate many other inflammatory molecules, such as E-selectin, vascular cell adhesion molecule-1, inducible nitric oxide synthase, and tissue factor, which are likely important in both RI and the eventual development of CGVD. These concerns are partially addressed by the complete reversal of increased RI after anti–ICAM-1 mAb administration. However, studies in ICAM-1 knockout mice point out the limitations of using anti–ICAM-1 mAb to support a role for its antigenic target in biologic processes. After intratracheal instillation of LPS, the development of pneumonia was successfully blocked by administration of anti–ICAM-1 mAb to wild-type mice compared with no difference from control in ICAM-1 mutants [29]. This disparity in the role of ICAM-1 when evaluated using mAb compared with mutant mice suggests that this "antigen-specific" inhibitor is exerting additional effects on endothelial, inflammatory, or myocardial cells other than blockade of ICAM-1. We therefore plan on using ICAM-1 antisense gene therapy to block ICAM-1 without concerns of nonspecific effects of mAb. Despite limitations, these results regarding the role of a donor systemic inflammatory state on early allograft ICAM-1 expression and severe RI with the eventual development of CGVD are convincing enough to warrant additional experiments in more clinically relevant large animal models.
In conclusion, donor exposure to an inflammatory stimulus (LPS), to simulate severe trauma and upregulate ICAM-1 before harvest, led to increased evidence of myocardial RI after global hypothermic ischemia and transplantation in our rodent model. The earlier and more intense expression at both the RNA and protein level during reperfusion in LPS-treated donors compared with healthy donors combined with the blockade of this increased RI in anti–ICAM-1 mAb-treated recipients supports the idea that ICAM-1 plays a central role. The combination of an increased incidence of acute rejection and more severe RI led to the development of an accelerated form of CGVD by day 90 after transplantation. Further studies are needed to directly demonstrate the significance of ICAM-1/neutrophil-mediated RI on primary cardiac dysfunction using functional parameters. This would allow a confirmation of increased RI without requiring sacrifice of the graft for later direct correlation with CGVD score, providing further support for their direct relationship. In addition, studies are underway to investigate the effects of blockade of this early RI on late graft outcome in this model.
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Acknowledgments |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Address reprint requests to Dr Robbins, Falk Cardiovascular Research Building, Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA 94305.
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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