Background: Rheumatoid arthritis (RA) is characterized by aggressive fibroblast-like synoviocytes (FLS) that mediate synovial hyperplasia, extracellular matrix (ECM) remodeling, and pannus formation. Recent studies have identified a subpopulation of pathogenic FLS within the synovial sublining layer marked by THY1 expression, which is expanded in RA and associated with local joint inflammation. Furthermore, THY1 ⁺ FLS interact with vascular endothelial cells through Notch signaling and contribute directly to pathological angiogenesis and pannus formation. Despite increasing evidence of FLS heterogeneity, the precise pathogenic FLS subsets and their upstream regulators responsible for synovial angiogenesis and pannus formation in RA are not yet clearly understood.

Objectives: In this study, we aimed to identify FLS subpopulations responsible for synovial hyperplasia, ECM remodeling and pannus development; elucidate the molecular mechanisms regulating their pathogenic activation; and explore therapeutic strategies targeting FLS activation and pannus-associated neovascularization in RA.

Methods: We integrated bulk RNA-sequencing, single-cell transcriptomics, and proteomics to identify pathogenic FLS subpopulations in RA synovium. Functional assays were conducted to assess the effects of AEBP1 and POSTN expression on FLS activation, ECM remodeling, and angiogenesis. We subsequently validated the regulatory function of TWIST1 in AEBP1 expression and investigated the pathological impact of TWIST1-AEBP1-POSTN axis in CIA mice model.

Results: We identified a pathogenic POSTN ⁺ FLS subgroup selectively enriched within the sublining layer of synovium that exhibits robust ECM remodeling and fibrogenic features. We demonstrated that AEBP1 is selectively elevated in these cells, where it activates TGF-β signaling to drive fibroblast activation, migration and proliferation. Besides, AEBP1 also induces POSTN expression, which are responsible for neovascularization. Further, TWIST1 directly regulates AEBP1 transcription in FLS. Intra-articular overexpression or knockdown of AEBP1 in CIA mice had opposite effects on synovial hyperplasia, bone erosion, and pannus formation. Moreover, the pharmacological inhibition of TWIST1 by harmine reduced AEBP1 and POSTN expression, limited the expansion of pathogenic THY1 ⁺ FLS, and alleviated joint pathology.

Conclusions: TWIST1 regulates AEBP1 expression in POSTN ⁺ FLS and amplifies RA pathology by sustaining TGF-β signaling. Disruption of the TWIST1–AEBP1–POSTN axis attenuates synovial hyperplasia and joint destruction, highlighting this pathway as a promising therapeutic target in RA.

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[2] Zhang, Y., J. Wang, H. Sun, et al., TWIST1+FAP+ fibroblasts in the pathogenesis of intestinal fibrosis in Crohn’s disease. J Clin Invest, 2024. 134(18).

[3] Croft, A.P., J. Campos, K. Jansen, et al., Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature, 2019. 570(7760): p. 246-251.

Acknowledgments: NIL.

Disclosure of Interests: None declared.