Background: Pediatric cholestatic liver diseases can be characterized by rapidly progressive fibrosis. A multicenter cross-sectional analysis of vibration-controlled elastography in biliary atresia (BA), alpha-1 antitrypsin deficiency (A1AT), and Alagille syndrome (ALGS) was leveraged to interrogate the plasma proteome relative to liver stiffness measurements (LSM). Methods: Slow off-rate modified aptamer scanning profiling of >7000 proteins in plasma from 187 children with BA (n=93), A1AT (n=31), ALGS (n=46), and healthy pediatric controls (n=17) was performed, and correlations with LSM were undertaken. Results: There was an abundance of LSM correlated proteins (BA n=2720, A1AT n=694, ALGS n=5968). Interestingly, a distinct plasma proteome was found in ALGS relative to BA and A1AT. Weighted Correlation Network Analysis identified groups of proteins with strong LSM correlation (eg, in a BA module of interest, Pearson correlation coefficient 0.79, p=5´0-21). Machine learning developed models predicting LSM as a continuous variable (median R ² =0.62 for BA). For BA, time to transplant could be predicted equally well by the proteome or clinical parameters (elastic net models achieved a C-index using proteome 0.91, clinical parameters 0.91, proteome and clinical parameters 0.90). Single-cell transcriptomics predicted the potential hepatic cell of origin for the most informative proteins, which included macrophage, mesenchymal, mesothelial, and endothelial cells. The epithelial-to-mesenchymal transition pathway was enriched in LSM correlated proteins in all 3 diseases. Conclusions: The plasma proteome is highly correlated in a disease-specific fashion with LSM in BA, A1AT, and ALGS. These correlations provide unique opportunities to identify biomarkers and focus attention on epithelial-to-mesenchymal transition in pediatric cholestasis.

Primary graft dysfunction (PGD) is a major cause of early morbidity and mortality after lung transplantation. During blood-based ex vivo lung perfusion (EVLP), hemolysis releases plasma-free hemoglobin (pfHb) and other erythrocyte-derived products that may promote endothelial injury and inflammation. We investigated whether EVLP-associated hemolysis is associated with cytokine release, endothelial dysfunction, and grade 3 PGD (PGD3). In this prospective, single-center observational study, end-EVLP perfusate from 57 double-lung transplant recipients supported with the Organ Care System Lung was analyzed for pfHb, 26 cytokines, blood counts, and associations with PGD3 within 72 h after transplantation. A mock EVLP blood-loop model was used to evaluate the effects of red blood cell storage duration and circuit run time on hemolysis. Generated hemolytic perfusates were applied to human lung microvascular endothelial cells to assess barrier injury and inflammatory activation. Among the 57 recipients, 32 (56.1%) developed PGD3 at any time point within 72 h after transplantation. Higher end-EVLP pfHb, interleukin-6, and interleukin-8 levels were associated with PGD3, and pfHb correlated positively with interleukin-6 and interleukin-8. Hemolysis increased with longer blood storage and EVLP duration, with storage duration emerging as the dominant contributor in the blood-loop model. Hemolytic perfusate disrupted endothelial junctions, increased permeability, and induced interleukin-6 and interleukin-8 release. EVLP-associated hemolysis is linked to endothelial barrier injury, inflammatory activation, and PGD3 after lung transplantation. Reducing hemolysis through improved blood-product selection and EVLP circuit management may represent a modifiable strategy to improve posttransplant outcomes.