Bioinformatics-driven single-cell sequencing analysis of angiogenesis-related genes in rheumatoid arthritis

To explore angiogenesis-related genes and immune cell profiles in RA and assess their clinical relevance in RA patients.

Gene expression datasets(GEO) of RA synovium were retrieved from the public database, and differential gene and immune infiltration analysis were performed using R. Angiogenesis-related genes were obtained from the Gene Set Enrichment Analysis(GSEA) database, and Receiver Operating Characteristic(ROC) curves were used to identify diagnostic genes based on their expression in RA synovium. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted using the DAVID and Metaspace database. Single-cell sequencing data related to RA were analyzed for dimensionality reduction and cell type annotation, with core genes identified. Finally, PBMCs from RA clinical samples were collected for Quantitative Real-Time PCR (RT-qPCR) and clinical correlation analysis.

This study identified 11 core genes associated with synovium and angiogenesis in RA. Notably, the diagnostic accuracy rates of IL10, E2F7, and E2F8 were found to be relatively high. Furthermore, we observed that these genes were significantly enriched in key signaling pathways such as HIF1 and VEGFA. Consequently, we determined that HIF1, VEGFA, E2F7, E2F8, and IL-10 are critical factors contributing to RA-related angiogenesis, with a significant correlation noted for the HIF1 signaling pathway. Additionally, our research indicates that various immune cell types, including B cells, plasma cells, T cells, natural killer (NK) cells, monocytes, macrophages, quiescent dendritic cells, mast cells, and neutrophils-are all implicated in the pathogenesis of RA. Relevant analyses demonstrate a positive correlation between HIF1 and IL10 as well as E2F7 and E2F8. However, the relationship between HIF1/VEGFA and common differential immune cell populations such as memory B cells, plasma cells, and activated memory CD4⁺ T cells is generally low. Further exploration of the clinical significance of the core target revealed that the mRNA levels of E2F7, E2F8, IL10, HIF1, and VEGFA in RA patients were significantly higher than those in the healthy control group. Among them, the levels of HIF1, VEGFA and E2F8 in patients with active RA were significantly higher than those in other periods, and their expression levels were positively correlated with the disease activity. In contrast, although the levels of E2F7 and IL10 in RA patients in remission decreased significantly, they were still higher than those in the normal control group.

The study pinpointed HIF1, VEGFA, E2F7, E2F8, and IL-10 as pivotal angiogenic genes whose expression tightly mirrors clinical disease activity. Notably, HIF1, VEGFA, and E2F8 escalate in parallel with increasing severity, are shaped by the inflammatory synovial milieu, and appear to fuel both angiogenesis and RA progression.