Single-cell analysis reveals fibroblast heterogeneity and myofibroblast conversion in ligamentum flavum hypertrophy

Background context: The ligamentum flavum (LF) is a crucial structure in maintaining spinal stability; however, hypertrophy of the LF is a significant contributor to lumbar spinal canal stenosis (LSCS). The mechanisms linking LF hypertrophy to the exacerbation of LSCS remain incompletely understood.

Purpose: This study aimed to investigate the cellular proportions and signaling pathways observed in the hypertrophied LF.

Study design: LF tissues were obtained from 3 patients undergoing lumbar decompressive surgery. These patients had been diagnosed with LSCS prior to surgery and had an LF thickness exceeding 3.5 mm.

Methods: Single-cell RNA sequencing was performed following LF tissue dissociation, and data were processed for quality control, dimensional reduction, and clustering. Differential gene expression and gene ontology analyses revealed key molecular pathways driving LF hypertrophy. Cell-cell communication analysis was analyzed to elucidate interactions among various cell types within the LF tissues.

Results: Fibroblasts accounted for 75% of the total cells, followed by endothelial cells, T cells, macrophages, and B cells. Among heterogeneous types of fibroblasts, we identified that a subset of fibroblasts trans-differentiated into myofibroblasts. Two types of macrophages that exhibited phenotypic plasticity akin to M1 and M2 states were observed. We also identified novel signaling pathways involved in fibroblast and immune cell interaction in the hypertrophied LF, such as GAS and GRN, as well as known signaling pathways, such as TGF-β, PDGF, CXCL, and ANGPTL.

Conclusion: Our study shows the changing cellular composition and pathogenic signaling pathways involved during the process of chronic inflammation highlighting the transdifferentiation process from fibroblasts to myofibroblasts in the hypertrophied LF.

Clinical significance: The identification of pathways such as GAS, GRN, TGF-β, ANGPTL, and CXCL, which appear to potentially contribute to LF hypertrophy, could significantly enhance our understanding of the pathogenesis of LSCS.