To determine the serum and tissue levels of fibroblast growth factor 2 (FGF2) in patients with Takayasu arteritis (TAK), and to investigate the mechanism by which FGF2-driven angiogenesis participates in the pathogenesis of TAK.

This study enrolled 48 patients with TAK from Beijing Anzhen Hospital and 24 age- and gender-matched healthy controls between March 2018 and March 2022. Serum levels of FGF2 and various cytokines were measured in all participants using the Bio-Plex Pro™ Human Cytokine 27-plex Assay kit. Furthermore, aortic tissue samples from 6 TAK patients and 6 patients with non-inflammatory aortic diseases, who underwent surgical treatment at Beijing Anzhen Hospital, between January 2023 and June 2024, were included for immunohistochemical and single-cell RNA sequencing (scRNA-seq). 

Significantly Upregulated FGF2 Levels in Serum and Aortic Tissues of the TAK Group

Serum FGF2 levels were significantly higher in the TAK group than in the control group [40.22 (29.88–60.01) pg/mL vs. 26.51 (21.28–30.08) pg/mL; p < 0.001] (Figure 1a), and higher serum FGF2 levels were associated with younger age at testing (p = 0.039) and earlier disease onset (p = 0.043) (Figure 1b), with a decreasing trend observed with advancing age. Besides FGF2, we measured a panel of pro-inflammatory cytokines, chemokines, and growth factors. Serum levels of G-CSF, MCP-1/CCL2, MIP-1α/CCL3, IL-4, IL-8, and IL-17 were significantly higher in TAK patients than in controls (all p < 0.001) (Figure 2a). In the TAK group (n = 48), serum FGF2 levels were positively correlated with multiple inflammatory mediators, including G-CSF, CCL2, CCL3, IL-4, IL-8, IL-17, IL-1β, IL-6, IL-12, IFN-γ, TNF-α, and VEGF (all p < 0.05) (Figure 2b). 

Immunohistochemical staining of aortic tissues from 3 TAK patients and 3 atherosclerosis patients showed significantly increased FGF2-positive cells in the TAK group (indicated by blue arrows, p = 0.011) (Figure 2c), which were mainly distributed around the neovascularized vasa vasorum in the aortic wall.

CD34hi_ECs Expansion and FGF2/FGFR1 Upregulation in TAK Aortic Tissues

After stringent quality control, 56,191 high-quality cells were retained for downstream analysis. Based on canonical markers from previous studies, the cells were clustered and annotated into 8 cell types: smooth muscle cells, T cells, macrophages, fibroblasts, endothelial cells, B cells, mesenchymal stem cells, and T cell-like smooth muscle cells (T cell-like SMCs) (Figure 3a). Endothelial cells identified by the markers PECAM1, FABP4, VWF, and IFI27 exhibited a markedly higher proportion (24.36% vs. 10.78%) and absolute number (6,572 vs. 3,150 cells) in the TAK group than in the control group (Figure 3b-c).

Analysis of aortic tissue scRNA-seq data identified fibroblasts, endothelial cells, and smooth muscle cells as the primary sources of FGF2 expression (Figure 3d). Next, we performed subpopulation analysis of endothelial cells using dimensionality reduction and clustering. All endothelial cells were divided into two clusters: one cluster with high CD34 expression was annotated as CD34-high endothelial cells (CD34hi_ECs), and the other was annotated as CD34-low endothelial cells (CD34lo_ECs) (Figure 3e). In the TAK group, 84.83% of the endothelial cells were identified as CD34hi_ECs, whereas in the control group, 95.71% of the endothelial cells were CD34lo_ECs, with only a minimal proportion being CD34hi_ECs (Figure 3i). We found that FGF2 expression was significantly higher in CD34hi_ECs than in CD34lo_ECs (Figure 3f). Moreover, GO (Gene Ontology) enrichment analysis revealed that CD34hi_ECs were mainly enriched in angiogenesis-related biological processes, including the regulation of apoptotic signaling pathways, regulation of vasculature development, and endothelium development (Figure 3h). Furthermore, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis indicated that CD34hi_ECs were significantly enriched in the MAPK signaling pathway (Figure 3h). We further analyzed the expression of FGF2 receptors, namely FGFR1 and FGFR2, across cell subsets. FGFR1 was predominantly expressed in fibroblasts, endothelial cells, and smooth muscle cells, with significantly higher expression in TAK group endothelial cells than controls (p < 0.001), while FGFR2 expression showed no difference between groups (p = 0.236) (Figure 3g). Furthermore, the expression of key pro-angiogenic genes in the FGF2/FGFR1 downstream MAPK (MAPK1, MAPK3, MAPK8) and PI3K (PIK3CA) pathways was significantly elevated in endothelial cells of the TAK group (Figure 3j).

FGF2 levels were significantly elevated in both the serum and aortic tissues of TAK patients compared with controls, and FGF2 was predominantly localized around the neovessels of the vasa vasorum in the vascular wall. Using scRNA-seq, we identified a CD34-high endothelial cell subset (CD34hi_ECs) that exhibited high FGF2 expression and enrichment in signaling pathways, including angiogenesis regulation and the MAPK pathway. We therefore hypothesize that CD34hi_ECs promote vasa vasorum neovascularization via the FGF2-FGFR1 autocrine/paracrine loop.