Abstract Body (Do not enter title and authors here): Background: After heart transplantation, endomyocardial biopsy is used to monitor for acute rejection (AR). However, endomyocardial biopsy is invasive, and its conventional histological interpretation has limitations. Extracellular vesicles (EVs) are lipid bilayer vesicles secreted by various cell types into the circulation. EVs express an array of biomolecules indicative of their parent cells and act as cargo vesicles capable of delivering nucleic acids, proteins, and organelles. Here, we investigated whether AR affects the surface markers and mitochondrial positivity of plasma EVs and explored their potential for detecting AR in cardiac transplant recipients.
Methods: Plasma samples were collected from participants from the Genomic Research Alliance for Transplantation (GRAfT, sponsored by NIH). Histopathology data were collected to define AR. Patients with non-rejection (NR, N=6), acute cellular rejection (ACR, N=7), antibody-mediated rejection (AMR, N=5), and a combination of ACR and AMR (ACR/AMR, N=2) were employed. Plasma EVs were isolated from a total of 71 plasma samples using a size exclusion column (qEV, IZON Science). The size distribution and concentration of the plasma EVs were evaluated by nanoparticle tracking analysis. The positivity for surface markers was assessed by flow cytometry, including CD235a (erythrocytes), CD144 (endothelial cells), CD45 (leukocytes), CD3 (T cells), CD20 (B cells), CD14 (monocytes), and CD172a (cardiomyocytes). Additionally, the positivity for mitochondrial membrane potentials in each EV cluster was measured.
Results: The concentrations and mean diameter of the plasma EVs were comparable among NR, AMR, and ACR groups (2.75 x 10¹⁰/mL, 9.61 x 10⁹/mL, and 2.70 x 10¹⁰/mL, and 192 nm and 211 nm, and 192 nm, respectively). The proportions of CD45 and CD172a-positive EVs were significantly increased in ACR/AMR compared to ACR or AMR alone. Additionally, mitochondrial markers within CD14-positive EVs exhibited a significant decrease in AMR. No significant trends were observed in the longitudinal EV profiles in each individual. Quantitative PCR confirmed that the expressions of mitochondrial DNA were decreased in the plasma EVs from AMR.
Conclusion: CD172a, CD45, and mitochondrial positivity in CD14-positive EVs could serve as potential biomarkers for diagnosing AR in patients undergoing cardiac transplantation. These findings may offer insights into the pathogenesis of AMR.