Section 2 -

Markers of Cardiac Allograft Injury Marlene L. Rose

Thoracic organ transplantation has enjoyed great success over the last decade; one year survival of patients receiving cardiac grafts in the most recent era (1999-2003) now strands at approximately 90%, the same as renal allografts [1]. Lung transplantation has improved in the last era (2000-2003) with one year survival being about 80% [2]. Despite the improvements in one year survival figures, long term allograftsurvival, of any organ, has not been impacted to the same degree. Thus, ten year survival with functioning grafts after heart and kidneys remains approximately 50%, with significantly less survival of allografted lungs at ten years. Loss of function of long-term cardiac allografts is due to onset of a gradual fibrotic obliterative disease affecting the donor venous and arterial structures, called cardiac allograft vasculopathy (CAV); histologically this consists of intimal hyperplasia, in the absence of an obvious vascular inflammatory infiltrate [3]. In contrast, lung pathology is characterised by fibrosis and obliteration of the airways [4]. The reasons for long term loss of graft function are due to immunological and non-immunological factors. There is interplay between the non-immunological factors (eg ischaemic reperfusion injury) and the adaptive immune response; for example, tissue injury results in upregulated expression of MHC and accessory molecules which will make donor cells more visible to the hosts' immune system. The process of apoptosis, a response to injury, results in expose of molecules, normally confined to intracellular spaces, on the outside of the cell, thus exposing autoantigens to the immune system. There is experimental evidence that allotransplantation breaks tolerance to self-antigens, which supports the emerging evidence for a role of autoimmunity in tissue damage after allotransplantation. One of the themes of this symposium is the concept that inflammatory tissue responses to injury are balanced by restorative responses. There is much evidence that cells and tissues have natural ways of cytoprotection and, we must assume that normally, restorative responses limit chronic tissue damage. Why this balance is not achieved after allotransplantation is incompletely understood, but may be related to the immune dysregulation which allows an autoimmune response to develop. The aim of this presentation is to describe markers of cardiac allograft damage, including autoimmune damage and recent evidence that demonstrates some natural protective mechanisms which become predominant in certain individuals. Acute rejection of cardiac allografts is diagnosed by the presence of an inflammatory mononuclear infiltrate in the endomyocardial biopsy and is graded according to international criteria [5]. Immunocytochemical analysis has revealed upregulation of MHC class I molecules on the myocardium [6] and induction or upregulation of various adhesion molecules (eg ICAM-1, VCAM-1) on graft endothelial cells [7]. These observations are consistent with the hypothesis that acute rejection is mediated by a T cells and monocytes secreting Th1 type pro-inflammatory cytokines. Of special interest, because it provides a link to the later complication of cardiac allograft vasculopathy (CAV), is the evidence of a hypercoaguable microcirculation, even in the first year after transplantation. This has been shown by an association between fibrin deposition and depletion of tissue-plasminogen activator from graft vasculature in patients who later develop CAV [8, 9]. The presence of C4d deposition in the micro-vessels, present in about 10% of surveillance cardiac biopsies, suggests a role for complement fixing anti-graft antibodies [10], also likely to be of direct damage to endothelial cell integrity. Anti-endothelial antibodies are formed in cardiac transplant recipients [11, 12, 13], and the presence of these antibodies is significantly correlated with the hypercoaguable state of the microcirculation [14] and development of CAV. Although western blots analysis was orginally used to detect anti-endothelial antibodies, 2-dimensional gel electrophoresis identified these antibodies as being to the autoantigen vimentin [15]; the same gels provide evidence for other autoimmune antibodies in serum of patients with CAV. Although, vimentin is well known as an intermediate filament, and as such it is not exposed to the immune system, recent papers have shown vimentin to be on the surface of apoptosed cells and platelets [16]. The fact that it on the surface of platelets, suggests a role of anti-vimentin antibodies and vimentin positive platelets in hypercoaguable graft endothelial cells and cardiac graft vasculopathy. This hypothesis is supported by the confocal imaging of human CAV lesions which demonstrates co-localisation of CD41 and vimentin and CD41, vimentin and PAI-1 (an inhibitor of fibrinolysis) within these lesions. Proteomics was also used to investigate markers of cytoprotection against CAV. Although common after cardiac transplantation, CAV is not inevitable. Our centre performed 2-dimensional gel electrophoresis of cardiac biopsies obtained from patients >9 years post-transplant with and without angiographic evidence of CAV. The most striking finding was a 20 fold increase in expression of hsp27 protein in cardiac biopsies of patients who were free of disease [17]. Mass spectrometry and other techniques demonstrated this to be a diphosphorylated from of hsp27. Confirmation was obtained by immunocytochemical analysis of paired biopsies from the same patients which demonstrated the hsp27 to be localised within blood vessels. In contrast, blood vessels in cardiac biopsies of patients with CAV lacked expression of hsp27. The possible vascular protective effects of hsp27 was also suggested by another proteomic study from this centre [18]. In this study proteomics was being used to detect changes in cardiac biopsies, associated with rejection, which might form the basis of a non-invasive test for acute rejection. Again, 2-dimensional gel electrophoresis was used to compare protein profiles from cardiac biopsies with and without histological evidence of rejection. The proteins alpha-crystalline and tropomyosin were found to be upregulated, and released into the circulation during acute rejection. Alpha crystalline is another small heat shock protein, similar to hsp27. The fact that it was being over-expressed during rejection, suggests the hypothesis that constant up-regulation of the small heat shock proteins is necessary to maintain healthy tissues. The hypothesis is that presence of these small heat shock proteins indicates healthy tissues and absence indicate disease. Data which supports this hypothesis has also been presented in the context of hsp27 and non-transplant atherosclerosis [19] and vascular disease associated with chronic renal allograft vasculopathy [20]. In conclusion, immunocytochemistry, confocal microscopy and proteomics have been used to determine the sequence of changes, in the graft following cardiac transplantation. The results demonstrate that an alloimmune response is not sufficient to explain the various outcomes after transplantation. A complex interplay between the innate immune system, regulation of autoimmunity and coagulation, and natural cytoprotective mechanisms can be demonstrated to play a part in graft outcome. 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