Blood Adv

Steroid-refractory acute graft-versus-host disease (SR-aGVHD) is a severe complication of allogeneic hematopoietic cell transplantation. While endothelial dysfunction is implicated in aGVHD pathophysiology, the status of the endothelium in patients with SR-aGVHD and the effect of the JAK1/2 inhibitor ruxolitinib at the microvascular level remain incompletely understood. This study aimed to characterize the endothelial damage phenotype induced by SR-aGVHD serum and to evaluate the potential modulatory effects of ruxolitinib. Human microvascular endothelial cells (HMEC-1) were exposed to serum from SR-aGVHD patients (n=21) or healthy donors (C), with or without ruxolitinib (0.4 M). Markers of endothelial activation (VCAM-1, ICAM-1), junctional integrity (VE-cadherin), prothrombotic state (VWF), apoptosis (cleaved caspase-3), and intracellular signalling (JAK-STAT and MAPK families) were analyzed by immunofluorescence and immunoblotting. Compared with C, SR-aGVHD serum caused increased VCAM-1 and ICAM-1 expression (p<0.05 for both), reduced VE-cadherin (p<0.01), elevated VWF production, and cleaved caspase-3 presence (p<0.05 for both). These changes were associated with significant phosphorylation of STAT3 (p<0.05), STAT1 (p<0.05), and key MAPK proteins (Erk1/2, p38, SAP/JNK, and cJun) (p<0.05, all). Ruxolitinib treatment effectively mitigated these responses, reducing adhesion molecule expression, restoring VE-cadherin localization, normalizing VWF production, and suppressing apoptosis. These protective effects were associated with a significant inhibition of the activated JAK-STAT and MAPK signalling pathways. SR-aGVHD serum directly induces endothelial activation in vitro, promoting a pro-inflammatory and prothrombotic phenotype. Ruxolitinib counteracts these effects, demonstrating a direct endothelial-protective role via suppression of JAK-STAT and MAPK signalling. These findings provide a novel mechanistic insight into its therapeutic efficacy in SR-aGVHD.