Trophic factor supplemented UW solution reduces intimal hyperplasia in the rat aortic transplant model

Methods F344 → F344 and Lewis → F344 orthotopic abdominal aortic transplants were performed after 48 h of cold storage in either UW or UW-TF solution with and without immunosuppression. Results Significant reduction in IH was observed when IG were stored in UW-TF solution compared to UW solution. A significant reduction in intimal inflammation was observed in UW-TF stored, nonimmunosuppressed AG. In immunosuppressed recipients, AG stored in UW-TF solution evidenced significantly less IH compared to those stored in UW alone. Conclusions UW-TF solution decreased IH in both alloindependent and dependent models. Keywords Intimal hyperplasia Rat aortic transplant model Trophic factors UW solution Chronic allograft nephropathy (CAN) is a major cause of premature renal allograft loss with attendant increases in recipient morbidity, health care costs, and demand on the donor organ supply. A number of risk factors have been identified for development of CAN and reduced graft survival after transplant. One of the most consistently cited significant factors for development of CAN is delayed graft function (DGF) [32,31,15] . The relationship between DGF and premature graft loss from CAN results from a complex interaction of both immunological and nonimmunological influences. Of the nonimmunologic influences cold ischemic injury has been consistently identified as one of the important donor-related risk factors for the occurrence of DGF, CAN, and premature graft loss [13,15,38,40,9,39,30] . With current organ storage methods, reduction in storage time has been the only feasible strategy identified for reduction of cold ischemic injury. In kidneys, CI periods greater than 24 h are significantly associated with reduced graft survival [30,35] . However, within the constraints of current clinical practices and organ sharing protocols it is frequently not possible to keep storage times below this threshold period. An alternative to reduced storage times is the reduction of cold ischemic injury by improvements in cold storage technologies [19–21,1,10,8,11] . This approach has recently gained momentum with the introduction of a new, modified UW solution containing bio-active factors, which has been shown to markedly reduce storage injury to kidneys in a canine autotransplant model as well as to liver in the porcine allograft model [19–21,1] . In these studies, graft damage was reduced by supplementation of UW solution with a unique combination of biologically active trophic factors, known to act in synergy, and which were shown to exert their effects during cold storage and not merely upon reperfusion [19] . The combination of trophic factors used in that study was subsequently optimized to a combination (substance P, bactinecin, nerve growth factor-β (NGF-β), insulin-like growth factor-1 (IGF-1)) that yielded further significant improvements in graft function even after long durations of cold storage [20,21] . The reduction in cold ischemic injury obtained with this improved solution holds promise for both reductions in DGF and reduced stimulus for development of CAN in the clinical transplant patient population. However, since the relative contributions of immune-dependent and nonimmunological influences to development of CAN in immunosuppressed allograft recipients remains controversial, it is unclear whether reductions in cold ischemic injury in this environment will substantially reduce development of the lesions associated with CAN. Functionally, CAN results in a progressive, irreversible decline in glomerular filtration ultimately resulting in premature graft loss. Histologically, CAN is characterized by tubular atrophy, interstitial fibrosis, and intimal hyperplasia (IH). IH, a critical lesion in CAN, reduces vessel lumen diameter and hence blood flow with subsequent ischemia to downstream tissues potentially augmenting tubular atrophy and interstitial fibrosis [28,29] . In the rat aorta transplant model, IH has been shown to result from both immunologic and nonimmunologic effects on endothelial cells in a manner similar to that seen in the human transplant patient population [44,22] . The objectives of this study were to determine the effect of select trophic factors added to the UW cold storage solution on development of cold ischemic injury-induced IH in aortic segments, studied in both the presence and absence of immunological influences after transplant. The hypothesis for this study is that grafts cold stored in UW solution with trophic factors will have less cold ischemic injury and therefore develop less IH, regardless of the immunological status of the graft relative to the recipient, compared to those stored in UW solution alone. Materials and methods Animals Male, approximately 300 g Fischer F344 and Lewis rats (Harlan, Indianapolis, Indiana) were used. F344 rats were used for isografting studies. Lewis rats were used as donors for allografts into F344 rats. All procedures and the use of these animals was reviewed and approved by the University of Wisconsin Institutional Animal Care and Use Committee. Procedure All rats were anesthetized with Isoflurane (Isoflo, Abbott Laboratories, North Chicago, IL) in 100% oxygen delivered via anesthetic mask. A midline abdominal incision was used to harvest a segment of abdominal aorta distal to the renal arteries and proximal to the aortic bifurcation. The graft was immediately flushed with cold (0–2 °C) unmodified UW solution [42] or cold UW solution supplemented with trophic factors (UW-TF). Trophic factors used to supplement the UW solution were as previously reported [20] and consisted of bactenecin, 1 mg/L (Multiple Peptide Systems, San Diego, CA); Substance P, 2.5 mg/L (Sigma, St. Louis, MO); IGF-1, 10 μg/L (Sigma, St. Louis, MO); and NGF-β, 20 μg/L (Research Diagnostics Inc., Flanders, NJ). Bactenecin was obtained by custom solid phase synthesis. IGF-1 and NGF-β were both human recombinant proteins produced in Escherichia coli . Grafts were cold stored on ice under sterile conditions in the same solution used for flushing the lumen for 48 h in plastic specimen containers. The containers had been previously conditioned by 12-h immersion in 10% bovine serum albumin, washed and then sterilized for use. After storage, the grafts were transplanted in an orthotopic position using an end-to-end anastomosis with 9–0 nylon suture in two simple continuous suture lines per anastomosis. Rats were recovered and given buprenorphine (0.05 mg/kg IM Q 12 h for 36 h as needed) (Buprenex, Reckitt Benckiser Healthcare (UK) Ltd., Hull, England) post-operatively for pain. Rats were euthanized immediately post-operatively if there was any evidence of hind limb weakness or cyanosis. Food and water were provided ad libitum. All rats analyzed in the study survived for 70 days and were euthanized by exsanguination under isoflurane anesthesia after harvest of the graft. Surgical controls were performed for both isografts and allografts. These aortic grafts were harvested, flushed with cold unmodified UW solution and immediately transplanted with minimal cold ischemic time (⩽1 h storage). Treatment groups Rats were divided into eight groups as follows ( Table 1 ): Group 1-isograft controls, Group 2-isografts stored in UW solution, Group 3-isografts stored in UW-TF solution, Group 4-allograft controls, Group 5-allografts stored in UW solution, Group 6-allografts stored in UW-TF solution, Group 7-allografts stored in UW solution with immunosuppressed recipients, Group 8-allografts stored in UW-TF solution with immunosuppressed recipients. Immunosuppresed recipients received oral cyclosporine-A (CsA) (10 mg/kg/day), a dose previously shown to suppress IH in this model in the absence of cold ischemic injury [16] . Cyclosporine treatment was begun two days prior to transplant and was continued until the time of euthanasia. Histomorphometric evaluation of IH in grafts Grafts were harvested after 70 days and divided transversely at the center of the graft. A section of recipient aorta proximal and distal to the graft was also harvested as control tissue. The aortic sections were stored in 10% neutral buffered formalin, processed routinely for histopathology, sectioned transversely and stained with hematoxyline and eosin. Grafts were embedded so that they were first sectioned from the center of the graft. Grafts were examined using light microscopy (Zeiss Axiovert 200M). Digital images were obtained of the transverse sections. Using image analysis software (Image-J 1.3V, National Institutes of Health, USA) the outer elastic lamina, the inner elastic lamina, and the endothelium were traced. The area of the intima was determined by subtracting the area of the vessel lumen from the area circumscribed by the inner elastic lamina. The area of the media was determined by subtracting the area circumscribed by the outer elastic lamina from the area circumscribed by the inner elastic lamina. Measurements from two sections from the center of each graft were made and averaged. The percent of the vessel wall that comprised the intima was also calculated [(intima)/(intima + media) × 100]. A pathologist, blinded as to the treatment groups, evaluated all grafts and graded the adventitia, media, and intima for inflammation. Inflammation scores ranged from 0 to 3. A score of 0, no inflammation; 1, mild, scattered cellular infiltration; 2, solid inflammatory infiltrate; 3, severe cellular infiltrate with disruption of vascular layers. Statistical method Data were analyzed for significant differences by analysis of variance (ANOVA) with pairwise comparisons performed using the Student–Newman–Keuls method. Non-normal data distributions were evaluated by Kruskal–Wallis ANOVA on Ranks with pairwise comparisons by Dunn’s method. Significant differences were determined at p < 0.05. Results Isografts In studies on isografts, 5 of 31 grafts were lost to analysis. In group 1 (surgical controls) 10 transplants were performed of which four experienced thrombosis of the graft and were euthanized immediately after surgery. In group 2 (UW isografts), nine transplants were performed of which one experienced thrombosis of the graft immediately post-operatively and was euthanized. In group 3 (UW-TF isografts), 12 transplants were performed and all survived. All grafts subjected to cold ischemic injury, regardless of the storage conditions, had significantly greater intimal areas compared to control isografts ( p < 0.01). However, grafts stored in UW-TF had 53% less CI-induced IH than grafts stored in unmodified UW ( p < 0.05) ( Table 1 ). The media area of UW stored isografts was 18% larger than isografts stored in UW-TF, however this difference was not statistically significant. There was significantly greater percent intimal area in isografts stored in unmodified UW solution (28.3 ± 5.2%) or UW-TF (20.3 ± 3.4%) compared to surgical controls (4.1 ± 1.1%) ( p < 0.01) ( Fig. 2 ). There was a trend toward reduction in percent intimal area for grafts stored in UW-TF solution compared to unmodified UW solution, however this difference was not statistically significant ( p = 0.055). There was no histologic evidence of inflammation observed in any isograft (i.e. all grafts were scored at 0 for inflammation). Nonimmunosuppressed allografts In group 4 (allograft controls), seven transplants were performed, one experienced graft thrombosis and was immediately euthanized. In group 5 (UW allografts), six transplants were performed and all survived. In group 6 (UW-TF allografts), seven transplants were performed and all survived. There were no significant differences in intimal area or media area between any groups in the nonimmunosuppressed allograft studies ( Table 1 ). Nor was there a significant difference in percent intimal area among allograft controls (28.1 ± 7.1%), allografts stored in unmodified UW solution (30 ± 4.4%), or allografts stored in UW-TF (41.4 ± 4.4%). Histologically allografts obtained from all groups in nonimmunosuppressed rats evidenced moderate to severe inflammation consisting of lymphocytes, plasma cells, histiocytes, and fibrosis in the adventitia, media, and endothelial cell layer ( Fig. 4 a). Quantitatively, however, there was significantly less inflammation observed in the intima of allografts stored in UW-TF (mean inflammation score = 0.57 ± 0.20) compared to allografts stored in unmodified UW solution (mean inflammation score = 1.67 ± 0.21) and surgical controls (mean inflammation score = 1.33 ± 0.21) ( Fig. 1 ) ( p < 0.05). Mean inflammation scores of the media were 0.83 ± 0.17, 1.00 ± 0.26, and 0.57 ± 0.20 for groups 4, 5, and 6, respectively. Mean inflammation scores of the adventitia were 2.67 ± 0.21, 2.5 ± 0.22, and 2.14 ± 0.14 for groups 4, 5, and 6, respectively. There was no significant difference in inflammatory scores of the media and adventitia between groups. Immunosuppressed allografts Group 7 (UW) had 15 transplants performed and all survived. Group 8 (UW-TF) had 11 transplants performed and all survived. In immunosuppressed recipients, there was 45% less IH induced by cold ischemic injury in allografts stored in UW-TF solution compared to those stored in unmodified UW ( p < 0.05), as well as significantly less percent intimal area (Group 7, 27.5 ± 2.4%; group 8, 19.0 ± 3.1%; p < 0.05) ( Fig. 3 ). The intimal areas of immunosuppressed allografts were statistically indistinguishable from isografts that had been stored under the same conditions. Histologically, in immunosuppressed allograft recipients, there was no to mild lymphoplasmacytic inflammation in the adventitia and was only occasionally observed in the endothelium ( Fig. 4 b and c). Mean inflammation scores of both the intimal and adventitial layers were 0.14 ± 0.1 and 0 for groups 7 and 8, respectively, whereas both groups had a mean inflammation score of 0 in the media layer. Both immunosuppressed groups had significantly less inflammation than the allograft controls ( p < 0.05), although no significant differences were noted in inflammation scoring between the two immunosuppressed groups or the immunosuppressed groups and the isograft groups ( Fig. 2 ). Discussion This study is the first to demonstrate that reductions in cold ischemic injury, without reducing cold ischemia time (CIT), can result in significant reductions in vascular intimal hypertrophy, even when studied in allografts in the presence of adequate immunosuppression. These results were achieved using trophic factors supplemented to cold UW storage solution, a modification of storage conditions which has been demonstrated in dog kidney autotransplants to markedly reduce cold ischemic injury and improve immediate graft function [19] . The beneficial effects of trophic factor supplementation have been shown to be obtained during the period of cold storage [19] . The ability of trophic factor supplementation to reduce cold ischemic injury-induced intimal hypertrophy was seen in both isografts and allografts. Isografts stored in UW-TF solution had 53% less ( p < 0.05) IH compared to grafts stored in UW solution. Similarly, allografts stored in UW-TF solution and transplanted into immunosuppressed recipients had 45% less ( p < 0.05) IH compared to those stored in unmodified UW solution. These findings further extend previous observations showing that preservation with appropriate trophic factors markedly improved immediate graft function and survival [20,21] by demonstrating that these effects also include a reduction in vascular lesions that are associated with degradative changes seen in CAN. Examination of existing reports shows that the cold ischemic time required for induction of vascular lesions is affected by the composition of the solution in which the grafts are stored [44,22,18,6,12,3,5,4] . Although no single study has systematically examined the impact of different preservation solutions on development of IH, these reports and others, when taken in aggregate, reinforce the concept that the quality of tissue preservation over time, with respect to suppression of cold ischemic injury and its initiating influence on development of graft vasculopathy, is critically affected by the specific composition of the solution used for cold storage. The addition of select trophic factors to the preservation solution significantly improved the quality of preservation obtained and may provide a platform for solution improvements in the future that either augment the effects of these trophic factors or suppress other key mechanisms not influenced by trophic factor activated pathways. The effect of immunosuppression on vascular lesions in rat aortic transplants has also been explored by a number of research groups [6,3,4,37,26,23,27,41,2,36] . In isograft aortic transplant models, it has been consistently shown that immunosuppression with CsA has no [6,41] or minimal [2] effects (likely secondary to CsA-induced smooth muscle toxicity) on cold ischemic injury-induced IH. Treatment of aortic isograft recipients with other immunosuppressive regimens including tacrolimus, mycophenolate mofetil, and a rapamycin analogue (SDZ RAD) has had variable effects on the development of IH induced by cold ischemic injury on isografted vessels [3,27] . This information suggests that there is little immunologic basis for IH formation in the isograft model; rather, the lesion is a direct result of nonimmunologic injury. Thus, it is clear from these reports and our current study that select trophic factor supplementation during cold storage must exert its effects via reductions in the alloindependent component of graft injury during cold storage. This view is supported by the significant reduction in IH observed in isografts stored in UW-TF solution compared to isografts stored in unmodified UW solution. Previous studies examining the development of IH in allografted aortas have used a variety of combinations of rat strains for donors and recipients. Regardless, IH in rat aortic allografts was uniformly more robust, inflammatory, and appeared earlier after transplant compared to results in isografts in these studies [44,12,3,26,23,41,36] , results clearly demonstrating that immune mediated mechanisms accelerate IH. Further, CsA has been shown to reduce or eliminate IH in rat aortic allografts when there was little to no cold ischemic injury [16,5,26,41,36] . However, when allografts are subjected to significant cold ischemic injury, immunosuppression with CsA or other immunosuppressive regimens does not prevent IH formation, furthering the hypothesis that both immune-independent and -dependent factors are co-involved in the pathogensis of IH and suggesting that formation of IH is a common end-point of several different injuries [4,23,5,37] . In this study, preservation of allografts with UW-TF solution did significantly reduce intimal inflammation in nonimmunosuppressed rats and resulted in significantly less IH in immunosuppressed allografts compared to allografts stored in UW. This may suggest a reduction of injurious allodependent factors, such as ischemia-induced expression of class II antigens, may have occurred in addition to reduction of the alloindependent factors (cold ischemic injury). Certainly, it is well known that cold ischemic injury will augment allogeneic-mediated injury in rat renal allografts [14] , and it is generally accepted that increased cellular damage causes increased antigenicity. Therefore, the reduction in cellular damage during cold ischemia may result in a reduction in antigenicity and a muted host immunologic reaction. It is significant that even in allografts without immunosuppression of the recipients, grafts stored in UW-TF solution had significantly lower inflammation scores in the intimal layer compared to both those stored in UW solution alone as well as the surgical allograft controls not subjected to cold ischemic injury. The pathogensis for IH in the aortic transplant model is unknown but is hypothesized to be similar to that of atherosclerosis, arising as a generic response to vessel injury [34] . Subsequent to injury, numerous growth factors and cytokines have been identified as being upregulated and are thought to participate in the formation of IH [12,43,7] . However, the role of exogenously administered growth factors in development or suppression of IH remains poorly understood. Interestingly, previous reports have shown that exogenous IGF-1, administered just prior to transplantation, accelerated rat aortic allograft arteriosclerosis [24,17,25] . However, the dose and context in which the grafts were exposed was significantly different than that used in our current study. In those grafts where IGF-1 induced a significant increase in IH, the amount administered was 50 times higher than was used in our studies to supplement UW solution. These contrasting results point out that the response to growth factors is highly dependent on the cellular context and concentration at which they are applied. It is well known that different results can be obtained at widely divergent concentrations of a given growth factor in individual cell types. Further, it is also known that the response to a growth factor is often modulated by the presence of other growth factors in the media. Thus, the actions seen with IGF-1 in our studies are likely advantageously modulated by the presence of the other trophic factors used in our trophic factor mixture. This supposition is supported by data generated in our laboratory in the dog kidney transplant model [20] and clearly a mechanism that should be elucidated in future studies. In this study, it should be noted that there appears to be a trend to an increase in intimal area in nonimmunosuppressed allografts stored in UW-TF (Group 7) compared to those stored in UW (Group 6), a difference that was not statistically significant. However, this apparent trend is spurious as it was shown, upon analysis of the data set, that one outlying data point accounted for more than 40% of the apparent variance and analysis after removal of this data point reduced the suggestion of a trend, decreased the variance in this group and even further substantiated that there was no significant difference compared to nonimmunosuppressed allografts stored in UW solution. Our working hypothesis is trophic factor supplementation reduces cold ischemic injury by preventing apoptosis as a result of trophic factor deprivation and/or supporting protective cell-signaling pathways during periods of hypothermia and ischemia. Recently, apoptosis of graft derived smooth muscle cells has been implicated in part of the pathophysiology of rat aortic intimal hyperplasia [33] . Prevention of the initiation of these apoptotic pathways may be a mechanism by which UW-TF reduces intimal hyperplasia, however this study was not designed to answer mechanistic questions but was targeted at identification of impacts of trophic factors on IH induced by cold ischemic injury. One flaw in our study design is the lack of a surgical control group for immunosuppressed allografts. The effects of CsA on IH in the aortic allograft model with no cold ischemic injury have been thoroughly documented [16,5,26,41,36] and our results in other experimental groups did not deviate from expected results based on current knowledge and literature. The exclusion of this control group does not impact the underlying findings of the study. The results of these experiments demonstrate that UW-TF solution has a direct positive effect on reducing both alloindependent and allodependent causes of IH in the rat aorta transplant model. These findings suggest that trophic factor supplementation may reduce the incidence of IH in allografts subjected to cold ischemic injury and therefore may reduce the incidence of CAN. Further work is needed to define the effect of TF supplementation to cold storage solutions on IH temporally and on various growth factors and cytokines known to be involved in graft vasculopathy. References [1] S. Ambiru K. Uryuhara S. Talpe Improved survival of orthopotic liver allograft in swine by addition of trophic factors to University of Wisconsin solution Transplantation 77 2004 302 319 [2] J.M. Bellón J. Buján F. Jurado A. Hernando N.G. Honduvilla B. Dominquez Cyclosporin A delays the presentation of intimal hyperplasia in an experimental model of arterial autograft Eur. Surg. Res. 28 1996 39 48 [3] O.J. Cole M. Shehata K.M. Rigg Effect of SDZ RAD on transplant arteriosclerosis in the rat aortic model Transplant. Proc. 30 1998 2200 2203 [4] O.J. Cole S.R. Stubington K.M. Rigg M. Shehata An investigation into the effects of cyclosporine and FK 506 on the progress of chronic rejection in the rat aortic model Transplant. Proc. 30 1998 991 [5] O.J. Cole K.M. Rigg M. Shehata The effect of different immunosuppressants on alloantigen dependent and independent factors involved in the development of chronic rejection in an animal model Ann. R. Coll. Surg. Engl. 83 2001 235 243 [6] J. Dominguez A. Kompatzki M. Schultz Effect of cyclosporine and ischemia on experimental transplant arteriosclerosis Transplant. Proc. 33 2001 3735 3736 [7] Y. Furukawa P. Libby J.L. Stinn G. Becker R.N. Mitchell Cold ischemia induces isograft arteriopathy, but does not augment allograft arteriopathy in non-immunosuppressed hosts Am. J. Pathol. 160 2002 1077 1087 [8] T. Hauet C. Tallineau J.M. Goujon M. Carretier M. Eugene J.P. Tillement Efficiency of trimetazidine in renal dysfunction secondary to cold ischemia-reperfusion injury: a proposed addition to University of Wisconsin solution Cyrobiology 37 1998 231 244 [9] G.R. Hetzel B. Klein M. Brause Risk factors for delayed graft function after renal transplantation and their significance for long-term clinical outcome Transpl. Int. 15 2002 10 16 [10] H. Huang Z. He L.J. Roberts A.K. Salahudeen Deferoxamine reduces cold-ischemic renal injury in a syngeneic kidney transplant model Am. J. Transplant. 3 2003 1531 1537 [11] A. Jani D. Ljubanovic S. Faubel J. Kim R. Mischak C.L. Edelstein Capase inhibition prevents the increase in capase-3, -2, -8, and –9 activity and apoptosis in the cold ischemic mouse kidney Am. J. Transplant. 4 2004 1246 1254 [12] R.J. Knight H. Liu E. Fishman E.D. Reis Cold ischemic injury, aortic allograft vasculopathy, and pro-inflammatory cytokine expression J. Surg. Res. 113 2003 201 207 [13] E.A. Kouwenhoven R.W. de Bruin U.W. Heeman R.L. Marquet J.N. Ijzerman Late graft dysfunction after prolonged cold ischemia of the donor kidney: inhibited by cyclosporine Transplantation 68 1999 1004 1010 [14] E.A. Kouwenhoven R.W. de Bruin I.M. Bajema R.L. Marquet J.N. Ijzermans Cold ischemia augments allogenic-mediated injury in rat kidney allografts Kidney Int. 59 2001 1142 1148 [15] N.R. Krieger B.N. Becker D.M. Heisey Chonic allograft nephropathy uniformly affects recipients of cadaveric, nonidentical living-related, and living-unrelated grafts Transplantation 75 2003 1677 1682 [16] D.M. Little R.L. Stoltenberg D.A. Hullett H.W. Sollinger Effect of neoral or cyclosporine on the development of chronic rejection in an aortic allograft rat model Transplant. Proc. 28 1996 880 881 [17] H. Lou Y. Zhao P. Delafontaine T. Kodama N.M. Katz M.L. Foegh Estogen effects on insulin-like growth factor-1 (IGF-1) induced cell proliferation and IGF-1 expression in native and allograft vessels Circulation 96 1997 927 933 [18] Y.P. Lu W.G. Chen I. Wang Y.P. Li A new rat model of transplant arteriosclerosis accelerated by ischemia/reperfusion injury Transplant. Proc. 35 2003 184 186 [19] J.F. McAnulty T.W. Reid K.R. Waller C.J. Murphy Successful six-day kidney preservation using trophic factor supplemented media and simple cold storage Am. J. Transplant. 2 2002 712 718 [20] J.F. McAnulty K.R. Waller M.K. Clayton C.J. Murphy Near total suppression of cold ischemic injury by an optimized trophic factor supplement to the UW preservation solution Am. J. Transplant. 3 2003 468 abstract [21] J.F. McAnulty J.D. Foley T.W. Reid T.D. Heath K.R. Waller C.J. Murphy Suppression of cold ischemic injury in stored kidneys by the antimicrobial peptide bactenecin Cyrobiology 49 2004 230 240 [22] A. Mennander S. Tiisala J. Halttunen S. Yilmaz T. Paavonen P. Häyry Chronic rejection in rat aortic allografts, an experimental model for transplant arteriosclerosis Arterioscler. Thromb. Vasc. Biol. 11 1991 671 680 [23] A. Mennander T. Paavonen P. Häyry Cylcosporine-induced endothelialitis and accelerated arteriosclerosis in chronic allograft rejection Transplant. Proc. 24 1992 341 [24] N. Motomura H. Lou P. Maurice M.L. Foeg Acceleration of arteriosclerosis of the rat aorta allograft by insulin growth factor-1 Transplantation 63 1997 932 936 [25] N. Motomura H. Lou H. Orskov P.W. Ramwell M.L. Foegh Exposure of vascular allografts to insulin-like growth factor-1 (IGF-1) increases vascular expression of IGF-ligand and receptor protein and accelerates arteriosclerosis in rats Transplantation 65 1998 1024 1030 [26] G.J. Murphy G.R. Bicknell M.L. Nicholson Microemulsion cyclosporin inhibits vascular remodeling and attenuates associated changes in profibrotic gene expression in an experimental model of allograft vasculopathy Br. J. Surg. 89 2002 1055 1061 [27] G.J. Murphy G.R. Bicknell M.L. Nicholson Rapamycin inhibits vascular remodeling in an experimental model of allograft vasculopathy and attenuates associated changes in fibrosis-associated gene expression J. Heart Lung Transplant. 22 2003 533 541 [28] B.J. Nankivell C.A. Fenton-Lee D.R.J. Kuypers Effect of histological damage on long-term kidney transplant outcome Transplantation 71 2001 515 523 [29] B.J. Nankivell R.J. Borrows C.L.S. Fung P.J. O’Connell R.D.M. Allen J.R. Chapman The natural history of chronic allograft nephropathy N. Engl. J. Med. 349 2003 2326 2333 [30] A.O. Ojo R.A. Wolfe P.J. Held F.K. Port R.L. Schmouder Delayed graft function: risk factors and implications for renal allograft survival Transplantation 63 1997 968 974 [31] L.C. Paul Pathophysiology of chronic renal allograft rejection Transplant. Proc. 1 1999 2715 2716 [32] O. Puyoo L. Rostaing E. Benard J.M. Cisterne A. Modesto D. Durand Risk factors for chronic rejection after renal transplantation: a retrospective study of 201 patients Transplant. Proc. 30 1998 2804 2806 [33] P. Religa K. Bojakowski M. Bojakowska Z. Gaciong J. Thyberg U. Hedin Allogenic immune response promotes the accumulation of host-derived smooth muscle cells in transplant arteriosclerosis Cardiovasc. Res. 65 2005 535 545 [34] R. Ross The pathogenesis of atherosclerosis: a perspective for the 1990’s Nature 362 1993 801 809 [35] A.K. Salahudeen N. Haider W. May Cold ischemia and reduced long-term survival of cadaveric renal allografts Kidney Int. 65 2004 713 718 [36] T. Schmitz-Rixen J. Megerman R.B. Colvin A.M. Williams W.M. Abbott Immunosuppressive treatment of aortic allografts J. Vasc. Surg. 7 1988 82 92 [37] H. Shimizu M. Takahasi S. Takeda Mycophenolate mofetil prevents transplant arteriosclerosis by direct inhibition of vascular smooth muscle cell proliferation Transplantation 77 2004 1661 1667 [38] D.A. Shoskes C.J. Michael Deleterious effects of delayed graft function in cadaveric renal transplant recipients independent of acute rejection Transplantation 66 1998 1697 1701 [39] J. Sienko M. Wisniewska M. Ostrowski Factors that impact on immediate graft function in patients after renal transplantation Transplant. Proc. 35 2003 2153 2154 [40] R. Sola A. Alarcon C. Jimenez A. Osuna The influence of delayed graft function Nephrol. Dial. Transplant. 19 2004 32 37 [41] R.L. Stoltenberg J. Geraghty D.M. Steele R. Kennedy D.A. Hullett H.A. Sollinger Inhibition of intimal hyperplasia in rat aortic allografts with cyclosporine Transplantation 60 1995 993 998 [42] J.A. Wahlberg J.H. Southard F.O. Belzer Development of a cold storage solution for pancreas preservation Cryobiology 23 1986 477 482 [43] J. Waltenberger M.L. Akyurek M. Aurivillius Ischemia induced transplant arteriosclerosis in the rat. Induction of peptide growth factor expression Arterioscler. Thomb. Vasc. Biol. 16 1996 1516 1523 [44] A. Wanders M.L. Akyürek J. Waltenberger Ischemia-induced transplant arteriosclerosis in the rat Arterioscler. Thromb. Vasc. Biol. 15 1995 145 155