Background/Purpose: Ankylosing spondylitis(AS) is a chronic inflammatory disease with clinical symptoms characterized by inflammatory low back pain and spinal ankylosis due to ligament ossification. Enthesitis is considered an important manifestation of ankylosing spondylitis and is involved in ligament ossification in AS. However, the exact mechanism of ligament ossification is still poorly understood. The aim of this study was to systematically investigate the mechanisms of AS ligament ossification through combination of multiple methods.

Methods: Single-cell sequencing of spinal ligament samples from AS patients (ligament ossification) and healthy controls. Pathway enrichment analysis (KEGG) and gene set enrichment analysis (GSEA) was conducted on the differential cell populations between the two groups. Eighteen-key differential genes (up-regulated in AS) identified by single cell sequencing were separately overexpressed in TT-D6 (mouse tendon cell line) using the CRISPR-Cas9 system. Intracellular glycosylation levels and mitochondrial membrane potential were detected by using a high-content cell imaging system and flow cytometry respectively. Effect of key gene overexpression on ligament ossification in vivo verified by tendon cell sheet transplantation experiments.

Results: Through single-cell transcriptome analysis, we identified a population of fibroblasts (ligament cells) with Prg4 as a marker gene whose proportion was significantly upregulated in ligament ossification AS patients (Figure 1A-C). Gene pathway enrichment analysis (KEGG) showed significant activation of glycolysis and nucleotide biosynthesis pathways in this subpopulation of cells in AS patients (Figure 1D). GFPT2 (Glutamine-Fructose-6-Phosphate Transaminse 2) as identified as a possible key gene involved in AS ligament ossification by a CRISPR-Cas9 gene screen combined with high content cell imaging system and flow cytometry (Figure 2A and 2B). In vivo transplantation experiments show that GFPT2 enhances abnormal ossification at the enthesis after patellar ligament resection modeling in mice (Figure 2C).

Conclusion: GFPT2 is a key gene for ligament ossification in AS, and targeting GFPT2 may serve as a novel approach to treating ankylosing spondylitis.

Figure1 A) Single-cell UMAP plot and cluster marker gene heatmaps of spinal ligament fibroblasts from healthy subjects, AS patients, and patients with degenerative spinal stenosis. B) Cluster composition map between three groups. C) Distribution of PRG4 and THY1 in different clusters. D) Pseudotime analysis of different modules. E) Gene pathway enrichment analysis (KEGG) of module 5 between healthy controls and AS patients.

Figure2 A) Detection of glycosylation levels in cells overexpressing different genes by high content imaging system using four different lectins. Mapping of heatmaps using high content parameters. B) Mitochondrial membrane potential (MPP) was detected by flow cytometry in cells overexpressing different genes. C) MicroCT assay of mice transplanted with different TT-D6 cell sheets after patellar ligament resection modeling. Red arrows indicate abnormal ossification at the enthesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/prg4-ligament-cells-are-involved-in-ligament-ossification-in-ankylosing-spondylitis/