Monika Matusiak-Brückner

Management of acute and especially chronic rejection after human cardiac transplantation is still challenging. Chronic rejection, represented by allograft vasculopathy (CAV) and cardiac interstitial fibrosis (CIF) is known to cause severe long-term complications. Rejection associated tissue-remodelling entails the reoccurrence of fetal variants of Fibronectin (Fn) and Tenascin-C (Tn-C), which are virtually absent in adult human organs. In a rat model, an extensive re-expression could be demonstrated for ED-A⁺ Fn with spatial association to CAV and CIF. Thus, it is of great interest to investigate the cardiac tissue expression and distribution in human samples. From 48 heart transplanted patients, 64 tissue specimens derived from right ventricular biopsies were available. Histopathological analysis was performed according to the International Society for Heart and Lung Transplantation (ISHLT) guidelines for the detection of acute rejection. By immunohistochemistry, protein expression of ED-A⁺ Fn, B⁺ Tn-C, alpha-smooth muscle actin, CD31 and CD45 was assessed and analysed semiquantitatively. Co-localisation studies were performed by means of immunofluorescence double labelling. Histopathological analysis of the 64 samples revealed different ISHLT grades (0R in 36 cases, 1R in 20 cases and 2R in 8 cases). There was a distinct and quantitatively relevant re-occurrence of ED-A⁺ Fn and B⁺ Tn-C in most samples. Semi-quantitative evaluation did not show any correlation to the acute rejection grade for all markers. Interestingly, significant correlations to the extent of inflammation could be shown for ED-A⁺ Fn (r = 0.442, p = 0.000) and B⁺ Tn-C (r = 0.408, p = 0.001) as well as between both proteins (r = 0.663, p = 0.000). A spatial association of ED-A⁺ Fn and B⁺ Tn-C to CAV and CIF could be demonstrated. A relevant re-occurrence of ED-A⁺ Fn and B⁺ Tn-C following human heart transplantation could be demonstrated with spatial association to signs of rejection and a significant correlation to tissue inflammation. These data might contribute to the identification of novel biomarkers reflecting the rejection process and to the development of promising strategies to image, prevent or treat cardiac rejection.

Objectives: Management of chronic cardiac rejection with allograft vasculopathy (CAV) and interstitial fibrosis (CIF) is still challenging. Rejection associated tissue remodelling entails the reoccurrence of fetal variants of Fibronectin (Fn) or Tenascin-C (Tn-C) that are virtually absent in adult human organs. In a rat model, ED-A⁺ Fn and B⁺ Tn-C could be shown to be extensively re-expressed in association to CAV and CIF. Since human antibodies recognizing the molecules and usable for targeted pharmacodelivery are available, it is of great interest to analyze ED-A⁺ Fn and B⁺ Tn-C expression also in the human system. Methods: Tissue specimens derived from right ventricular biopsies from 69 heart transplant recipients were available. Histopathological examination to identify acute rejection was performed according to the ISHLT guidelines. Protein expression of ED-A⁺ Fn, B⁺ Tn-C, alpha-smooth muscle actin (ASMA) and CD31 was analysed by immunohistochemistry / immunofluorescence and subjected to semi-quantitative assessment or co-localization analysis respectively. Results: Histopathological analysis of the 69 samples revealed ISHLT grade 0R in 36 cases, grade 1R in 21 cases and grade 2R in 8 cases. There was a relevant re-expression of ED-A⁺ Fn and B⁺ Tn-C in the great majority of cases. Semi-quantitative analysis did not show any correlation to ISHLT grade for all three markers. But there was a significant positive correlation between expression of ED-A⁺ Fn and B⁺ Tn-C (p = 0.000). There was a spatial association of ED-A⁺ Fn and B⁺ Tn-C to vessel structures and fibrosis as revealed by immunofluorescence double labelling including ASMA and CD31. Conclusions: There is a relevant re-occurrence of ED-A⁺ Fn and B⁺ Tn-C following human cardiac transplantation contributing to extracellular matrix reorganization as an initial process of tissue remodelling. Against the background of a correlation of ED-A⁺ Fn and B⁺ Tn-C expression to chronic rejection in animal models, one can postulate that patients with an early re-occurrence of the markers are at an increased risk to develop CAV and CIF. Moreover, the association of ED-A⁺ Fn and B⁺ Tn-C deposition to signs of chronic rejection and the existence of antibodies for targeted delivery of diagnostics or bioactive payloads, might pave the way for novel innovative strategies to image, prevent or treat the disease.