Background: The role of Programmed cell death 1 ligand 1 (PD-L1) in regulating cellular stress response beyond its classical role in cancer-immunology is increasingly recognized. Emerging evidence indicates a central role in cardiac disease, but its regulation, subcellular trafficking and its dynamics as circulating PD-L1 remain poorly understood. As a translational approach, we explored the regulation and spatial distribution of PD-L1 in coronary endothelial cells exposed to hypoxia/reoxygenation (H/R) and evaluated soluble PD-L1 in patients with acute myocardial infarction (AMI).
Methods: Human coronary artery endothelial cells (HCAECs) were exposed to hypoxia (1 % O₂, 20 h) followed by reoxygenation for 4, 24, 48, and 72 h. PD-L1 expression was analyzed using western blot and flow cytometry. Super-resolution microscopy was applied to visualize subcellular localization and spatial distribution of PD-L1. PD-L1 in endothelial supernatants and isolated exosomes were determined by enzyme-linked immunosorbent assay (ELISA). Bulk RNA sequencing was performed to characterize global transcriptional responses. As a translational approach, PD-L1 was quantified in serum samples of patients with acute myocardial infarction (AMI) and non-AMI controls and correlated with clinical characteristics. Statistical analysis was conducted using t-test or Mann-Whitney U test for non-normally distributed values; Chi2 test was applied for binary data.
Results: H/R induced a strong increase in overall endothelial PD-L1 after 4 h (1.58 ± 0.13-fold, p < 0.001), which persisted until 72 h (1.43 ± 0.19-fold, p = 0.036), mainly driven by increased membrane-bound PD-L1 at 24 h onward (24 h: 1.48 ± 0.06-fold, p < 0.001; 72 h: 1.58 ± 0.11-fold, p < 0.001), as shown by flow cytometry. Super-resolution microscopy revealed redistribution of PD-L1 along the cell membrane within vesicular structures, starting at 24 h and persisting up to 72 h, accompanied by its release into the extracellular space. RNA sequencing demonstrated activation of inflammatory signaling pathways together with changes in genes related to vesicular transport and secretion. Involved pathways included nuclear factor-κB (NF-κB) signaling and Ras-related protein Rab-11A (Rab11A)-mediated vesicular trafficking, coordinating PD-L1 induction and secretion. Exosomal PD-L1 tended to rise after 4 h of reoxygenation (1.91 ± 0.36-fold, p = 0.051) and remained elevated at 48 h (1.74 ± 0.10-fold, p < 0.001). For the translational analysis, 13 AMI patients and 41 non-AMI controls were included. Corresponding to the in-vitro findings, soluble PD-L1 was higher in AMI patients compared to controls (AMI: 139.05 ± 146.28 pg/ml; non-AMI: 14.35 ± 28.40 pg/ml; p = 0.013).
Conclusion: HCAECs showed a dynamic change in PD-L1 expression during H/R, accompanied by spatial redistribution and enhanced exosomal release. RNA sequencing indicated involvement of inflammatory and vesicle-associated pathways in PD-L1 regulation. The presence of elevated soluble PD-L1 in patient serum following AMI may reflect similar endothelial stress responses in-vivo. These findings provide experimental and clinical evidence supporting endothelial PD-L1 as an immunomodulatory target in AMI.
