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Cold Ischemia Induces Isograft Arteriopathy, but Does Not Augment Allograft Arteriopathy in Non-Immunosuppressed Hosts

2002; Elsevier BV; Volume: 160; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)64928-0

1525-2191

Yutaka Furukawa, Peter Libby, Jennifer L. Stinn, Gerold Becker, Richard N. Mitchell,

Organ Transplantation Techniques and Outcomes

Prolonged cold ischemia has been suggested as a factor that will exacerbate later graft arterial disease (GAD), a major limiting factor for long-term transplant survival. We therefore examined the effects of cold ischemia on GAD as well as adhesion molecule and cytokine expression in murine cardiac grafts. Mild GAD developed in isografts undergoing 4-hour cold ischemia. Relative to control isografts, cold ischemia induced transiently enhanced endothelial expression of intercellular adhesion molecule-1 (ICAM-1) at 4 hours post-transplant. There was also transiently-augmented gene expression of interleukin (IL)−1β, IL-6, and transforming growth factor-β in these cold-ischemic isografts. By 3 days post-transplantation, however, there were no longer any differences between control and cold ischemic isografts. Cold ischemia did not significantly affect the final grade of either parenchymal rejection or GAD in long-term (4 to 12 weeks) major histocompatibility complex (MHC) I- or MHC II-mismatched allografts molecules transplanted without immunosuppression. At early time points after cold ischemia (4 to 24 hours), allografts mismatched for MHC I and/or MHC II showed enhanced expression of ICAM-1 and cytokines comparable to that seen in isografts. By day 7 post-transplant, both control and cold ischemia allografts showed comparable expression of cytokines and adhesion molecules. Although prolonged cold ischemia can initiate mild GAD in isografts by transiently enhancing antigen non-specific inflammatory responses, it does not significantly augment subsequent alloresponses. Prolonged cold ischemia has been suggested as a factor that will exacerbate later graft arterial disease (GAD), a major limiting factor for long-term transplant survival. We therefore examined the effects of cold ischemia on GAD as well as adhesion molecule and cytokine expression in murine cardiac grafts. Mild GAD developed in isografts undergoing 4-hour cold ischemia. Relative to control isografts, cold ischemia induced transiently enhanced endothelial expression of intercellular adhesion molecule-1 (ICAM-1) at 4 hours post-transplant. There was also transiently-augmented gene expression of interleukin (IL)−1β, IL-6, and transforming growth factor-β in these cold-ischemic isografts. By 3 days post-transplantation, however, there were no longer any differences between control and cold ischemic isografts. Cold ischemia did not significantly affect the final grade of either parenchymal rejection or GAD in long-term (4 to 12 weeks) major histocompatibility complex (MHC) I- or MHC II-mismatched allografts molecules transplanted without immunosuppression. At early time points after cold ischemia (4 to 24 hours), allografts mismatched for MHC I and/or MHC II showed enhanced expression of ICAM-1 and cytokines comparable to that seen in isografts. By day 7 post-transplant, both control and cold ischemia allografts showed comparable expression of cytokines and adhesion molecules. Although prolonged cold ischemia can initiate mild GAD in isografts by transiently enhancing antigen non-specific inflammatory responses, it does not significantly augment subsequent alloresponses. Progress in immunosuppressive therapy and management of acute allograft rejection has improved short-term survival of heart transplant patients. However, strategies for prevention and treatment of graft coronary artery disease (GAD) have proven elusive and GAD remains a major limiting factor for long-term graft survival.1Uretsky BF Murali S Reddy PS Rabin B Lee A Griffith BP Hardesty RL Trento A Bahnson HT Development of coronary artery disease in cardiac transplant patients receiving immunosuppressive therapy with cyclosporine and predonisone.Circulation. 1987; 76: 827-834Crossref PubMed Scopus (479) Google Scholar, 2Paul LC Fellstrom B Chronic vascular rejection of the heart and the kidney; have rational treatment options emerged?.Transplantation. 1992; 53: 1169-1179Crossref PubMed Scopus (137) Google Scholar Various immunological, infectious, and nonimmunologic factors may contribute to the development of GAD.3Gao SZ Hunt SA Schroeder JS Alderman EL Hill IR Stinson EB Early development of accelerated graft coronary artery disease: risk factors and course.J Am Coll Cardiol. 1996; 28: 673-679Abstract Full Text PDF PubMed Google Scholar, 4Hornick P Smith J Pomerance A Mitchell A Banner N Rose M Yacoub M Influence of acute rejection episodes, HLA matching, and donor/recipient phenotype on the development of "early" transplant-associated coronary artery disease.Circulation. 1997; 96: 148-153PubMed Google Scholar, 5Weis M von Scheidt W Cardiac allograft vasculopathy: a review.Circulation. 1997; 96: 2069-2077Crossref PubMed Scopus (401) Google Scholar, 6Johnson MR Transplant coronary disease: nonimmunologic risk factors.J Heart Lung Transplant. 1992; 11: S124-S132PubMed Google Scholar Characteristically, GAD affects the engrafted vessels and spares the host arteries. Although understanding of the pathogenesis of GAD is incomplete, two simple models can explain this selective involvement of the transplanted arteries: an immunological response directed against donor antigens, or a response to ischemic injury encountered during storage and transport postexplantation.7Day JD Rayburn BK Gaudin PB Baldwin 3rd, WM Lowenstein CJ Kasper EK Baughman KL Baumgartner WA Hutchins GM Hruban RH Cardiac allograft vasculopathy: the central pathogenetic role of ischemia-induced endothelial cell injury.J Heart Lung Transplant. 1995; 14: S142-S149PubMed Google Scholar Thus, Gaudin et al showed that histologically proven ischemic injury during the perioperative period predicts the development of GAD in humans.8Gaudin PB Rayburn BK Hutchins GM Kasper EK Baughman KL Goodman SN Lecks LE Baumgartner WA Hruban RH Peritransplant injury to the myocardium associated with the development of accelerated arteriosclerosis in heart transplant recipients.Am J Surg Pathol. 1994; 18: 338-346Crossref PubMed Scopus (73) Google Scholar Another recent study9Schmid C Heemann U Tilney NL Factors contributing to the development of chronic rejection in heterotopic rat heart transplantation.Transplantation. 1997; 64: 222-228Crossref PubMed Scopus (55) Google Scholar demonstrated the development of GAD in rat heart isografts following prolonged cold ischemia. The mechanisms for the development of GAD by cold ischemia/reperfusion are not fully understood. Several studies have demonstrated that warm ischemia and reperfusion resulted in increased cell adhesion molecule expression and stimulated reactive oxygen species and inflammatory cytokine production in a variety of organs, culminating in leukocyte accumulation and tissue destruction.10Kukielka GL Hawkins HK Michael L Manning AM Youker K Lane C Entman ML Smith CW Anderson DC Regulation of intercellular adhesion molecule-1 (ICAM-1) in ischemic and reperfused canine myocardium.J Clin Invest. 1993; 92: 1504-1516Crossref PubMed Scopus (214) Google Scholar, 11Wyble CW Desai TR Clark ET Hynes KL Gewertz BL Physiologic concentrations of TNF-α and IL-1β released from reperfused human intestine up-regulate E-selectin and ICAM-1.J Surg Res. 1996; 63: 333-338Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 12Colletti LM Remick DG Burtch GD Kunkel SL Strieter RM Campbell DA Role of tumor necrosis factor-α in the pathophysiologic alterations after hepatic ischemia-reperfusion injury in the rat.J Clin Invest. 1990; 85: 1936-1943Crossref PubMed Scopus (762) Google Scholar Much less is known about the effects of cold ischemia and reperfusion on early cytokine expression. It also remains controversial whether prolonged cold ischemia/reperfusion injury can aggravate GAD.9Schmid C Heemann U Tilney NL Factors contributing to the development of chronic rejection in heterotopic rat heart transplantation.Transplantation. 1997; 64: 222-228Crossref PubMed Scopus (55) Google Scholar, 13Knight RJ Dikman S Liu H Martinelli GP Cold ischemic injury accelerates the progression to chronic rejection in a rat cardiac allograft model.Transplantation. 1997; 64: 1102-1107Crossref PubMed Scopus (69) Google Scholar Particularly, it is uncertain whether early enhanced inflammation induced by prolonged cold ischemia can accentuate subsequent alloimmune responses, or whether ischemic injury and alloimmune responses may independently affect the development of GAD. The present study used a heterotopic mouse heart transplant model to examine whether cold ischemia followed by reperfusion can induce GAD in isografts not subject to immunological injury, or augment GAD in major histocompatibility complex (MHC) I- or MHC II-mismatched allografts. We chose a four-hour ischemic period to correspond to the upper limit of cold ischemia typically permitted for clinical human heart transplantation. To gain mechanistic insight into the pathogenesis of transplantation complications, we further studied the effects of prolonged cold ischemia on the time course and magnitude of expression of inflammatory cytokines and cell adhesion molecules in isografts and in MHC I-, MHC II-, or in total-allomismatched allografts. The results indicate that cold ischemia transiently increases the expression of selected cytokines, as well as intercellular adhesion molecule-1 (ICAM-1), and may thereby contribute to the development of GAD. However, alloresponses in cardiac grafts occur largely after the effects of ischemic injury have already subsided, and the extent of acute parenchymal rejection or subsequent GAD are not significantly affected by prior cold ischemic injury. Antibodies for mouse ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and isotype- and class-matched immunoglobulin were purchased from PharMingen (San Diego, CA). Anti-mouse P-selectin antibody (10A10) was a generous gift of Dr. Michael A. Gimbrone, Jr. Normal goat serum, rabbit serum, and biotinylated secondary antibodies were from Vector Laboratories Inc. (Burlingame, CA) [α-32P]UTP was from Perkin Elmer Life Sciences, Inc. (Boston, MA). Inbred male mice (9 to 12 weeks) of several strains were used in these experiments. C57BL/6 (B/6, H-2b) and BALB/c (B/c, H-2d) mice were obtained from Taconic Farms, Inc. (Germantown, NY). B6-C-H-2bm12KhEg (bm12, H-2bm12) mice MHC class II-mismatched from B/6 mice, and B6-C-H-2bm1ByJ (bm1, H-2bm1) mice MHC class I-mismatched from B/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained in the Harvard Medical School animal facilities and allowed ad libitum access to food and acidified water. Sentinel mice housed in the same room as experimental animals were consistently pathogen-free. All experiments conformed to animal care protocols approved by the institutional review group. Heterotopic heart transplantation was performed as previously described.14Corry RJ Winn HJ Russell PS Primary vascularized allografts of hearts in mice.Transplantation. 1973; 16: 344-350Crossref Scopus (777) Google Scholar, 15Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (229) Google Scholar In brief, donor and recipient mice were anesthetized by inhalation of Metofan (Pittman-Moore, Mundelein, IL). Donor hearts were perfused with chilled, heparinized 0.9% saline or heparinized Stanford solution16Drinkwater DC Rudis E Laks H Ziv E Marino J Stein D Ardehali A Aharon A Moriguchi J Kobashigawa J University of Wisconsin solution versus Stanford cardioplegic solution and the development of cardiac allograft vasculopathy.J Heart Lung Transplant. 1995; 14: 891-896PubMed Google Scholar via the inferior vena cava and harvested after ligation of the vena cava and pulmonary veins. The aorta and pulmonary artery of donor hearts were anastomosed to the abdominal aorta and inferior vena cava of recipient mice, respectively, using microsurgical technique. Ischemic time during the surgical procedure was routinely 30 minutes, and initial graft survival was greater than 90%. For cold ischemia experiments, harvested donor hearts were perfused and stored in sterile saline (0.9% NaCl solution) for 4 hours at 4°C before transplantation. Preliminary experiments showed minimal histological evidence of myocardial necrosis at day 7 post-transplant in B/6 heart isografts following 4-hour cold ischemia in saline (data not shown). Different storage solutions may potentially offset the effects of prolonged cold ischemia on GAD. We therefore also tested the effects of cold ischemia on GAD development in the presence of Stanford solution, a storage medium reported efficacious for reducing GAD in human heart transplantation.16Drinkwater DC Rudis E Laks H Ziv E Marino J Stein D Ardehali A Aharon A Moriguchi J Kobashigawa J University of Wisconsin solution versus Stanford cardioplegic solution and the development of cardiac allograft vasculopathy.J Heart Lung Transplant. 1995; 14: 891-896PubMed Google Scholar In this series of experiments, we evaluated GAD in MHC II- disparate grafts at 8 weeks and in MHC I-disparate grafts at 12 weeks. Stanford solution experiments, including non-ischemic controls, were performed as a distinct cohort from the saline experiments. Grafts were explanted at defined intervals of 0 hours, 4 hours, 24 hours, 3 days, 7 days, or 4, 8, or 12 weeks post-transplant, and sectioned into three transverse parts. The basal third was fixed in 10% phosphate-buffered formalin, embedded in paraffin, and 5 μ sections were stained with hematoxylin and eosin, or elastic tissue stains. The other transverse sections were either frozen in OCT compound (Ames Co., Division of Miles Laboratories, Elkhart, IN) for immunohistochemistry (see below) and/or analyzed by RNase protection assay (RPA; see below). The severity of parenchymal rejection and GAD was scored blindly by two independent observers (Y.F. and R.N.M.); scores uniformly fell within a range of one grade for the two observers, and were averaged. Parenchymal rejection was graded using a scale modified from the International Society for Heart and Lung Transplantation17Billingham ME Cary NR Hammond ME Kemnitz J Marboe C McCallister HA Snovar DC Winters GL Zerbe A A working formation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart rejection study group.J Heart Lung Transplant. 1990; 9: 587-593Google Scholar (0, no rejection; 1, mild interstitial or perivascular infiltrate without necrosis; 2, focal interstitial or perivascular infiltrate with necrosis; 3, multifocal interstitial or perivascular infiltrate with necrosis; and 4, widespread infiltrate with hemorrhage and/or vasculitis). The severity of GAD was evaluated based on the lumenal narrowing by intimal hyperplasia, and was scored individually for each vessel (0, no or minimal [ 10 vessels on three to four transverse sections were averaged for each specimen. Preliminary experiments demonstrated that GAD develops more rapidly in MHC II-disparate grafts relative to MHC I-disparate grafts, frequently exhibiting complete lumenal occlusion by 8 weeks. Conversely, post-transplant intervals of 12 weeks are typically required to develop moderate-to-severe GAD in MHC I-disparate grafts. Because the composition of cellularity versus matrix deposition in GAD varies as the lesions mature,15Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (229) Google Scholar we evaluated GAD at two time points. MHC II-disparate grafts were therefore assessed at 4 and 8 weeks post-transplant, while MHC I-disparate grafts were examined at 8 and 12 weeks. The expression of cell adhesion molecules following immunohistochemical staining was evaluated blindly by two observers (Y.F. and R.N.M): −, not present or focal weak staining; +, diffuse or strong staining; these evaluations were 100% concordant. Total RNA was prepared by guanidinium thiocyanate/phenol/chloroform/isoamylalcohol isolation method using TRIZOL (Gibco BRL). Fifteen micrograms of total RNA for each sample was analyzed quantitatively for cytokine mRNA expression by RPA. Multiprobe RNase protection assay kit mCK-3b (containing DNA templates for tumor necrosis factor (TNF)-β, lymphotoxin (LT)-β, TNF-α, interleukin (IL)−6, interferon (IFN)-γ, IFN-β, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, macrophage migration-inhibitory factor (MIF), L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) and mCK-2b (containing DNA templates for IL-12p35, IL-12p40, IL-10, IL-1α, IL-1β, IL-1 receptor antagonist (Ra), IL-18, IL-6, IFN-γ, MIF, L32, and GAPDH) were obtained from PharMingen. RPA was performed following the manufacturer's instructions. In brief, the DNA templates were used to synthesize the [α-32P]UTP-labeled RNA probes in the presence of GCAU mixture and T7 RNA polymerase. Each RNA sample was hybridized with the labeled RNA probes by overnight incubation at 56°C, followed by digestion with RNase A and T1 mixture for 45 minutes at 30°C. The samples were treated by proteinase K, extracted with Tris-saturated chloroform-isoamylalcohol-phenol solution, and then ethanol-precipitated in the presence of ammonium acetate. The protected RNA duplexes were dried, redissolved in loading buffer and, after denaturing at 90°C, resolved on a 5% acrylamide-urea sequencing gel. 32P-labeled probes were used as molecular size markers. The gel was absorbed to filter paper, dried under vacuum, and exposed on film (BioMax- MR; Kodak, Rochester, NY) and phosphorimager screen. The volume integrations of mRNA-protected32P-labeled probe fragments were analyzed by bioimaging analyzer (Molecular Dynamics, Sunnyvale, CA). Finally, volume integrations of the protected bands for each cytokine were normalized against the bands for GAPDH in the corresponding lane. Total RNA samples from naive hearts of donor strain were used as normal heart controls. Four-μm-thick frozen sections were fixed in acetone for 5 minutes at 4°C. To reduce nonspecific binding, the sections were first incubated with 5% normal goat or rabbit serum for 30 minutes at room temperature (RT). Hamster monoclonal antibody against mouse ICAM-1 and rat monoclonal antibodies against mouse VCAM-1, P-selectin, and E-selectin were used as primary antibodies. Sections were incubated with primary antibodies for 90 minutes at RT, washed in phosphate buffered saline (PBS), and incubated with biotinylated secondary antibodies (goat anti-hamster IgG[H + L] or rabbit anti-rat IgG[H + L]) for 45 minutes at RT. After washing in PBS, the sections were incubated with an alkaline phosphatase-conjugated avidin-biotin complex (Vectastain ABC-AP kit; Vector Labs) and washed. Alkaline phosphatase activity was visualized by incubating in substrate solution (Fast Red; Sigma Chemical Co., St. Louis, MO). Sections were counterstained with hematoxylin. Naive hearts of the donor strain were used as normal heart samples. Sections of mouse placenta were used for positive control staining of P-selectin and E-selectin. The incidence of GAD in isografts was compared between groups using χ2Paul LC Fellstrom B Chronic vascular rejection of the heart and the kidney; have rational treatment options emerged?.Transplantation. 1992; 53: 1169-1179Crossref PubMed Scopus (137) Google Scholar test. Values for histological grading of parenchymal rejection and GAD in allografts, and values for relative gene expression of inflammatory cytokines were expressed as mean ± SEM and compared using analysis of variance (ANOVA) followed by Fisher's protected least significant difference (PLSD) post-hoc test. The majority of arteriopathy lesions in isografts were mild, with an average GAD score for each graft of less than 1. Therefore, to evaluate the effects of prolonged cold ischemia on GAD in isografts, we elected to compare the incidence of GAD between no-storage controls and 4-hour cold ischemia groups. Mild intimal thickening was observed by 8 weeks in the coronary arteries of isografts which had undergone 4-hour cold storage in saline (Figure 1B), whereas isografts of the no-storage group did not show any histological features of GAD at 8 weeks (Figure 1, A and C). The incidence of low-grade GAD in cold-ischemic isografts tended to increase at later time points (12 weeks), although there was no statistically significant difference in the incidence of GAD between no-storage group and 4-hour cold storage group at that time point. Notably, well-developed intimal lesions with severe luminal narrowing were seen only in the 4-hour cold ischemia group at 12 weeks (Figure 1D). Four hours of cold ischemia in Stanford preservation solution tended to increase the extent of GAD relative to storage in saline, but the incidence of GAD was not significantly different at 12 weeks between Stanford solution and saline groups (Table 1).Table 1Incidence of GAD in IsograftsTime after transplantation4 weeks8 weeks12 weeksSaline storage No storage0 (n = 4)0 (n = 4)17 (n = 6) 4-hour cold ischemia0 (n = 5)50 (n = 4)60 (n = 5)Stanford solution No storageNDND25 (n = 4) 4-hour cold ischemiaNDND80 (n = 5)Values are expressed as percent incidence of GAD. All of the averaged GAD scores applied for isografts were less than grade 1. Note the greater values of incidence of GAD (indicated by bold letters) in 4-hour cold ischemia groups relative to the no storage groups.ND, not done. Open table in a new tab Values are expressed as percent incidence of GAD. All of the averaged GAD scores applied for isografts were less than grade 1. Note the greater values of incidence of GAD (indicated by bold letters) in 4-hour cold ischemia groups relative to the no storage groups. ND, not done. Both MHC I- (bm1 to B/6) and MHC II-mismatched (bm12 to B/6) allografts develop mild to severe GAD by 8 to 12 weeks post-transplant (Figure 1). Because totally allomismatched (B/c to B/6) allografts undergo severe acute rejection and cease functioning at approximately 8 days,18Nagano H Libby P Taylor MK Hasegawa S Stinn JL Becker G Tilney NL Mitchell RN Coronary arteriosclerosis after T-cell-mediated injury in transplanted mouse hearts: role of interferon-γ.Am J Pathol. 1998; 152: 1187-1197PubMed Google Scholar they do not survive long enough to develop GAD in the absence of immunosuppression. Thus, B/c to B/6 allografts were not evaluated for GAD. Four-hour cold storage in either 0.9% saline or Stanford preservation solution did not significantly exacerbate or accelerate the development of GAD for either MHC I or MHC II antigen-mismatched allografts, compared with corresponding no-storage control groups (Table 2, Table 3). Although the use of Stanford solution for donor heart perfusion and cold preservation resulted in significantly greater parenchymal rejection and GAD scores relative to grafts perfused in 0.9% saline (Table 3), the Stanford and saline solution groups were transplanted as distinct cohorts at separate times, and are not strictly comparable. Inflammatory cell infiltration accompanied by more widespread myocyte necrosis (parenchymal rejection) occurred by day 3 in all allografts and became more prominent by day 7. In both MHC I- and MHC II-mismatched allografts, parenchymal rejection diminished at later stages (by 4 to 8 weeks), whereas GAD progressed by 4 to 8 weeks.15Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (229) Google Scholar Prolonged cold ischemia did not significantly affect the severity nor change the time course of parenchymal rejection in any of the allograft combinations (Table 2, Table 3).Table 2GAD and Parenchymal Rejection Score in Allografts Stored in Saline4 weeks8 weeks12 weeksTime after transplantationGADPRGADPRGADPRbm1 → B/6 No storageNDND1.21 ± 0.43 (7)1.93 ± 0.30 (7)0.93 ± 0.40 (7)1.63 ± 0.39 (8) 4-hour cold ischemiaNDND1.92 ± 0.57 (6)1.86 ± 0.26 (7)0.64 ± 0.21 (7)*p < 0.05 vs. 4-hour cold ischemia (bm1 to B/6) allograft group at 8 weeks.1.07 ± 0.13 (7)bm12 → B/6 No storage1.33 ± 0.63 (6)1.75 ± 0.50 (6)1.36 ± 0.51 (7)2.71 ± 0.26 (7)NDND 4-hour cold ischemia1.43 ± 0.58 (7)2.29 ± 0.53 (7)1.43 ± 0.40 (7)2.43 ± 0.23 (7)NDNDValues are expressed as mean ± SEM. PR, parenchymal rejection; GAD, graft coronary artery disease. The scales (1–4) are described in the Methods.ND, not done.* p < 0.05 vs. 4-hour cold ischemia (bm1 to B/6) allograft group at 8 weeks. Open table in a new tab Table 3GAD and Parenchymal Rejection Score in Allografts Stored in Stanford Solution8 weeks12 weeksTime after transplantationGADPRGADPRbm1 → B/6 No storageNDND1.43 ± 0.47 (7)2.00 ± 0.19 (7) 4-hour cold ischemiaNDND2.42 ± 0.52 (6)1.93 ± 0.37 (7)bm12 → B/6 No storage2.86 ± 0.21 (7)3.57 ± 0.17 (7)NDND 4-hour cold ischemia3.08 ± 0.54 (6)3.25 ± 0.21 (6)NDNDValues are expressed as mean ± SEM.PR, parenchymal rejection; GAD, graft coronary artery disease. There was no statistically significant difference in GAD and parenchymal rejection scores between no storage and 4-hour cold ischemia Stanford solution groups in each donor strain. Significantly greater values (p < 0.05) than those of the corresponding saline groups (see Table) are indicated by underlining.ND, not done. Open table in a new tab Values are expressed as mean ± SEM. PR, parenchymal rejection; GAD, graft coronary artery disease. The scales (1–4) are described in the Methods. ND, not done. Values are expressed as mean ± SEM. PR, parenchymal rejection; GAD, graft coronary artery disease. There was no statistically significant difference in GAD and parenchymal rejection scores between no storage and 4-hour cold ischemia Stanford solution groups in each donor strain. Significantly greater values (p < 0.05) than those of the corresponding saline groups (see Table) are indicated by underlining. ND, not done. Figure 2 shows representative RPA gels from mRNA derived from isografts or total mismatched allografts; the associated densitometric analyses of these gels are shown in Figure 3. Normal hearts contained minimal levels of mRNAs encoding inflammatory cytokines such as IL-6 and IL-1β. However, expression of the mRNAs for these cytokines increased by 4 hours after transplantation in all grafts. This rise is attributable to the transplantation surgery which entails an obligatory 30 minutes of warm ischemia. Cold ischemia increased expression of IL-6 and IL-1β mRNAs in all donor and recipient strain combinations tested. TGF-β mRNA was expressed weakly in normal hearts, and increased in all grafts within 24 hours of transplantation. Cold ischemia (4 hours) enhanced this increase in TGF-β by 24 hour post-transplantation, reaching statistically significant differences between no-storage group and 4-hour cold ischemia group in TGF-β1, β2 and β3 in isografts, and in TGF-β2 and β3 in allografts. In isografts, TGF-β1 and β2 mRNA decreased by day 7 and there was no significant difference between the no-storage and 4-hour cold ischemia groups. Thus, in isografts, the effects of prolonged cold ischemia (relative to no-storage) on cytokine mRNA expression are evident only at early time points (1 day) after transplant.Figure 3The results of densitometric analysis of cytokine mRNA expression. Volume integrations of the mRNA bands for each cytokine were analyzed using phosphoimaging analyzer and normalized against the bands for GAPDH in the corresponding lane. Graphs showing the relative densitometric values of IL-1β, IL-6, TNF-α, TGF-β1, TGF-β2, TGF- β3, IFN-γ, or IL-10 mRNA expression in isografts and MHC I-disparate (bm1 to B/6), MHC II-disparate (bm12 to B/6), or totally mismatched (B/c to B/6) allografts (from left to right). Three to four animals per each group were analyzed. Each relative densitometric value is expressed as mean ± SEM. *P < 0.05 vs no storage group at the same time point, †P < 0.05 vs each corresponding group at day 3. Open bars, no storage control groups; hatched bars, 4-hour cold ischemia groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT) However, in allografts, TGF-β1 remained elevated at day 7, while TGF-β2 and β3 tended to decrease. Beginning at day 7 post-transplant, mRNAs encoding the T cell cytokine IFN-γ and another proinflammatory cytokine TNF-α were markedly increased in all allografts and, particularly in total allomismatched allografts; transcripts for IL-1β, IL-6, TGF-β1, and IL-10 were also strongly expressed. In contrast to the impact of prolonged cold ischemia on the immediate expression of IL-6, IL-1β, and TGF-β in isografts, there were no differences between the 4-hour cold ischemia group and the control no-storage group in the levels of IL-10 or IFN-γ mRNA at early time points (<3 days). Moreover, there were no significant differences between the 4-hour cold ischemia group and the control no-storage group in the mRNA transcripts of any cytokines measured beginning with the peak of mRNA expression at day 7. The temporal changes of T cell cytokine expression were similar for MHC I, MHC II, or total alloge