Study Objective and Hypothesis: The pathogenesis of fibrosis during metabolic dysfunction-associated steatohepatitis (MASH) is not fully understood. Our previous work indicate that the Eph/Ephrin signaling could be a major cell-cell signaling hub regulating fibrogenesis in MASH. While cell-cell interaction between HSC and liver sinusoidal endothelial cells (LSEC) has long been proposed as a driver for liver fibrosis, molecular mechanisms of their interaction have not been elaborated. In this study, we tested the hypothesis that EphrinB ligands expressed in LSEC plays a role in the pathogenesis of MASH fibrosis. Methods: Single nuclear RNA-sequencing of liver from human and mouse MASH were analyzed for Efnb1 and Efnb2 expression. Human liver tissues obtained from patients with MASH were assessed for pEphrinB expression. Plasma level of EphrinB2 was measured in MASH and healthy individuals. EphrinB1 deletion in LSEC were generated by crossing Efnb1fl/fl mice with CDH5(PAC)-CreERT2 mice. Experimental MASH was developed in LSEC-specific Efnb1 knockout and wild type mice by feeding them with a choline-deficient amino-acid defined (CDAA) high fat diet for 8 weeks. Histology, flow cytometry, qPCR, biochemical assays and immunohistochemistry were used to dissect the contribution of LSEC- Efnb1 function in MASH. Statistical analysis was performed using ANOVA and Mann Whitney U test and a p-value < 0.05 was considered significant. Data: Single nuclear RNA sequencing showed that Efnb1 and Efnb2 are upregulated in LSEC of mice with MASH. Plasma level of Ephrin-B2 is significantly elevated in MASH patients compared to healthy individuals. Deletion of Efnb1 was achieved after tamoxifen administration, as depicted by reduced expression of Efnb1 mRNA level and EphrinB1 protein in the liver of LSEC Efnb1−/− mice compared to littermate ( Efnb1fl/fl) fed the CDAA-HFD. However, we noted an increased expression of Ephb receptors mRNA level in the liver of LSEC Efnb1−/− mice compared to littermate fed the CDAA-HFD. In addition, we noted an increased CDAA-HFD-mediated inflammation and fibrosis after deletion of Efnb1 in LSEC compared to littermates mice fed CDAA-HFD. Conclusions: Although EphrinB1 is upregulated in LSEC during advanced MASH, its deletion does not protect from MASH fibrosis. Given the functional redundancy between Ephrin-B ligands, deletion of one member of this family might not be suffcient to abolished EphB-EphrinB bidirectional signaling. NIDDK, NIAMS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.