Heterogeneity of Active Mast Cells, Endothelial Cells, and Fibroblasts: Unique Transcriptomic Features of Synovial Single-Cell Sequencing in Hemophilic Arthritis

Purposes Recurrent synovial bleeding is the major cause of Hemophilic Arthritis (HA). Up to now, the transcriptomic profiles of bleeding syvonium in HA patient have largely remained unknown. Exploring the transcriptomic characteristics of synovium obtained from HA patients undergoing total knee arthroplasty (TKA), and uncovering potential pathological mechanisms of HA synovium through bioinformatics analysis and differential analysis. Methods Single-cell RNA sequencing (scRNA-seq) technology was utilized to identify distinct cellular subsets within HA synovium. Comparative analysis was conducted with scRNA-seq data from osteoarthritis (OA) and rheumatoid arthritis (RA) synovium to assess transcriptional differences. Histological evaluation, immunofluorescence (IF), immunohistochemistry (IHC) and in vitro cell testing were performed for validation. Results We observed that the single-cell transcriptomic characteristics of HA synovium differ significantly from those of OA and RA patients. Mast cells, identified as unique immune cells in HA synovium, are actively involved and may be the initiating factor for changes in vascular permeability or bleeding in HA synovium. The transcriptomic features of endothelial cells and fibroblasts, which are non-immune stromal cells, reveal the synovial microenvironment: repeated bleeding leads to iron deposition, which causes substantial ferroptosis within the synovium. Based on the pseudotime analysis of endothelial cells, along with IHC staining and in vitro cell assays, it was demonstrated that ferroptosis in endothelial cells induces vascular damage and triggers a significant migration of endothelial cells. The stress of ferroptosis leads to the differentiation of HMOX1+ endothelial cells. Cell interaction analysis shows that HMOX1+ endothelial cells can induce the SCARA5+ fibroblast subtypes via FTL/SCARA5. This may be a critical target for alleviating cellular ferroptosis in HA synovium. Conclusions Through scRNA-seq, we elucidated the heterogeneity of synovial tissues in three diseases. Activated mast cells around blood vessels may be potential target cells for bleeding and rebleeding, with TPSB2 emerging as a potential target. We also identified endothelial cell subpopulations under ferroptosis stress and their interactions with fibroblasts. Notably, we discovered a SCARA5+ fibroblast subpopulation that regulates ferroptosis, which could serve as a potential target gene for preventing and treating recurrent bleeding in HA. Adjust the cell concentration to the ideal range of 700-1200 cells/μL. Gel beads containing barcode information are mixed with cells and enzymes. Then encapsulated in oil surfactant droplets within a microfluidic system, forming Gel Beads-In-Emulsions (GEMs). GEMs flow into a collection reservoir where gel beads dissolve, releasing barcode sequences, reverse transcribing cDNA fragments, and labeling the samples. Gel beads are ruptured and oil droplets shattered to PCR amplify cDNA using it as a template. The products from all GEMs are mixed to construct a standard sequencing library. Initially, cDNA is enzymatically fragmented into segments around 200-300 bp, followed by traditional second-generation sequencing library construction processes like adding sequencing adapters and primers, and finally PCR amplification to obtain a DNA library. High-throughput sequencing of the prepared library is performed using Illumina sequencing platform's paired-end sequencing mode. After obtaining raw data, the official analysis software Cell Ranger from 10x Genomics (https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome) is used for data filtering, alignment, quantification, cell identification, and obtaining gene expression matrices for each cell.