Gkantsinikoudi, C.; Dignam, J. P.; Kumar, R.; Jokl, E. J.; Li, W.; Samus, M.; Landi, S.; Athwal, V.; Kendall, T. J.; Rot, A.; Fallowfield, J. A.; Piper Hanley, K.; Alazawi, W.; Dufton, N. P.

Vascular plasticity is a crucial biological asset enabling our bodies to rapidly adapt to infections and acute inflammation. However, repeated insult during chronic disease can result in these vascular adaptations becoming irreversible, thereby driving disease progression and fibrosis. This study aimed to understand if phenotypic changes in endothelial cell (EC) identity could be indicative of progressive fibrosis and thereby offer new diagnostic and therapeutic opportunities for patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Previous research has documented that a significant shift in EC transcriptomic signature occurs during liver fibrosis in both pre-clinical models and patients. However, the protein expression profile, phenotype and functional role of these new EC subpopulations that are induced during fibrogenesis is unclear. In this study, we integrate high-resolution imaging, proteomic and transcriptomic analysis which collectively highlight a central role for endothelial-to-mesenchymal transition (EndMT)-induced EC plasticity in the derivation of fibrosis-associated EC (FAEC). We demonstrate that: 1) full spectrum flow cytometry can provide new opportunities to categorize and phenotype EC subpopulations, 2) two distinct EndMT-derived FAEC subpopulations expand during fibrogenesis; THY1.2+ICAM1+ and TAGLN+MCAM+ EC that display unique immunomodulatory and metabolic phenotypes, 3) TAGLN+ FAEC are a conserved, pro-fibrotic cell type arising at early stages of MASLD, and 4) increased hepatic expression of TAGLN is significantly associated with detrimental patient outcomes at all stages of liver disease. This study will pave the way for the development of FAEC-specific diagnostic and therapeutic approaches to tackle progressive fibrotic disease.