P6345Chronic cardiac allograft rejection after heterotopic rat heart transplantation: ed-a but not ed-b domain containing fibronectin as novel serum biomarker of cardiac allograft vasculopathy
Authors: Franz M , Rossberg M , Gruen K , Baez L , Schulze P C , Berndt A 
Published: October 2019

Abstract

Background and aims
Diagnosis of Cardiac Allograft Vasculopathy (CAV) following heart transplantation (HTX) is still challenging but of prognostic relevance. Thus, the identification of biomarkers non-invasively detecting the disease is an hitherto unmet clinical need. Fetal variants of the cell adhesion modulating protein Fibronectin (Fn) could be proven to re-occur in the context of chronic rejection in rat and human cardiac tissue after HTX in spatial association to CAV and fibrosis. This study was aimed to determine serum levels of fetal Fn variants in a heterotopic rat HTX model of chronic rejection.

Methods
Chronic cardiac allograft rejection was induced by heterotopic rat HTX using inbred male Lewis (LEW) rats as donors and Fisher (F344) rats as recipients (n=24). After microsurgical modifications, the allograft was implanted into the abdomen of the recipient animal. Rats were sacrificed after 70 days. Untreated F344 rats were used as controls (n=6). Cardiac tissue was subjected to histological analysis according to a recently established sum-score system between 0 and 10 (0 = no detectable rejection; 1 to 3 = weak rejection; 4 to 7 = moderate rejection; and 8 to 10 = severe rejection). The score includes all relevant parameters of chronic rejection (CAV, lymphocyte infiltration, cardiomyocyte degeneration, fibrosis). Brain natriuretic peptide (BNP) was determined by a commercially available ELISA. Serum levels of both, ED-A domain containing Fn (ED-A+ Fn) and ED-B domain containing Fn (ED-B+ Fn) were quantified using ELISA protocols established in our group.

Results
Final experiments were performed on day 70, mortality rate was 0%. Histological analysis revealed a chronic cardiac allograft rejection in all transplanted animals (mean sum-score 6.92±1.94) while controls did not exhibit relevant alterations (mean sum-score 0.1±0.2; p<0.001). There were significantly elevated BNP levels in HTX rats (76.16±23.22 pg/ml) compared to controls (352.23±132.09 pg/ml; p<0.001). BNP showed a strong positive correlation to the histological sum-score (r=0.653; p<0.001). Serum levels of ED-A+ Fn (HTX: 34.42±11.4 μg/ml, controls: 17.3±6.13 μg/ml; p=0.009) but not ED-B+ Fn (HTX: 8.58±4.12 μg/ml, controls: 9.4±2.61 μg/ml; p=0.804) were significantly increased in HTX rats qualifying ED-A+ Fn for further analysis with respect to histological signs of chronic rejection. Interestingly, there is a noticeable association to the occurrence of CAV representing 1 out of 4 parameters included in the histological sum-score (r=0.51; p=0.006).

Conclusions
Chronic cardiac allograft rejection and, in particular, CAV as major long-term prognosis-limiting factor after HTX are accompanied by both, a re-expression and vessel associated tissue deposition of ED-A+ Fn and also a relevant liberation into blood. These results underline the potential impact of ED-A+ Fn as biomarker or potential therapeutic target patients that underwent HTX.