Endothelial and inflammatory responses during ex vivo normothermic perfusion of human cardiac grafts

AIMS: Normothermic ex vivo heart perfusion (NEVHP) allows functional assessment and preservation of donor hearts, but the biological responses occurring during perfusion are not well characterized. This pilot study evaluated the feasibility of sequential biomarker monitoring during human cardiac NEVHP and described inflammatory, endothelial, and hemostatic responses over time. METHODS: This single-center, prospective, observational pilot study included five consecutive donor hearts preserved with the TransMedics Organ Care System (OCS) at the University Hospital of Rennes between March 2021 and January 2023. OCS use was indicated for expected cold ischemia >4 hours or surgical complexity. Perfusate samples were collected after priming (T0), at 10 minutes (T1), 60 minutes (T2), and before cooling (T3). Cytokines, chemokines, endothelial markers, and hemostatic factors were quantified by multiplex immunoassay and ELISA. Data were analyzed using linear mixed-effects models and expressed as fold-change versus T0. RESULTS: Donor median age was 44 years (IQR 36-50) and 60% male. All grafts were transplanted. Recipient mean age was 53±9 years and 20% female. Two of five (40%) developed severe primary graft dysfunction. Sequential sampling was successful in all perfusions. Inflammatory mediators rose during perfusion: at T3 versus T0, IL-8 increased 11.5-fold (95% CI 6.5-20.1), bFGF 8.4-fold (6.7-10.5), IL-6 4.5-fold (2.4-8.2), and MCP-1/CCL2 4.1-fold (2.6-6.6). IL-10 and TNF-α showed smaller increases (2.0- and 1.6-fold). Leukocyte counts remained stable (0.57×109/L at T0; fold-change 0.9). Endothelial markers showed activation without evidence of injury. Angiopoietin-2 increased 1.6-fold (1.2-2.1) and VEGF 2.0-fold (1.2-3.5), while angiopoietin-1, syndecan-1, soluble E-selectin, thrombomodulin, VEGFR2, PlGF, and vWF:Ag showed minimal or inconsistent changes. These trajectories are consistent with endothelial activation in the absence of glycocalyx shedding or structural disruption. Despite high heparin levels (median 6.9 IU/mL), low-grade hemostatic activation occurred. D-dimer increased 1.9-fold (1.3-2.7), fibrin monomer 2.2-fold (1.2-3.9), and soluble P-selectin 1.5-fold (1.1-2.0). Platelet counts declined to 0.8 (0.7-0.9) relative to baseline. Hematocrit decreased slightly (15.5% to 14.6%, fold-change 0.94), consistent with mild hemodilution. CONCLUSIONS: Sequential biomarker monitoring during NEVHP was feasible and demonstrated inflammatory, endothelial, and hemostatic changes. These biological patterns require confirmation in larger cohorts, as potential tools for graft assessment and optimization of perfusion circuits and perfusate composition.