To investigate the regulatory role of injured vascular endothelial cells in the fibrosis progression in the necrotic region of the femoral head in a glucocorticoid-induced osteonecrosis of the femoral head (GONFH), and further elucidate the underlying molecular mechanisms, so as to provide a theoretical basis for anti-fibrotic intervention and clinical treatment of GONFH.

① Clinical sample histological examination: Femoral head samples were collected from 5 patients diagnosed with GONFH admitted to Department of Orthopedics of Daping Hospital between December 2023 and December 2025 and assigned to the experimental group. Meanwhile, femoral head specimens from 5 patients undergoing surgical treatment for femoral neck fracture (FNF) were selected as the normal control group. Paraffin sections from the reactive zone of necrotic tissues were prepared. HE staining, Sirius red staining, Western blotting for α-smooth muscle actin (α-SMA) and fibronectin (FN), and CD31 immunofluorescence staining were performed to evaluate tissue morphology, collagen deposition, expression of fibrosis-related proteins and distribution of vascular endothelial cells. ② In vitro experiments: The effect of factors secreted by dexamethasone (DEX)-treated human umbilical vein endothelial cells (HUVEC) on myofibroblast activation was determined. Human bone marrow mesenchymal stem cell (hBMSC) was induced to differentiate into myofibroblasts using transforming growth factor-β (TGF-β). HUVEC were then divided into a DEX treatment group (48 h) and a PBS control group, and conditioned medium (CM) was collected from both groups. CM-DEX and CM-PBS were co-incubated with myofibroblasts for 48 h, and Western blotting was applied to detect the protein expression levels of α-SMA, collagen type Ⅰ (Col Ⅰ), and FN in myofibroblasts. ③ Exploration of molecular mechanisms: Total RNA was extracted from DEX-treated and control HUVEC for RNA sequencing (RNA-seq) to screen differentially expressed genes (DEGs) between the 2 groups. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. Secreted proteins from both groups of HUVEC were collected for proteomic sequencing, and differentially secreted proteins were screened. Bioinformatics analysis was used to analyze their pathways and association with myofibroblast activation.

① Clinical sample findings: Compared with the FNF control group, the reactive zone of the femoral head in the GONFH group showed: disorganized tissue arrangement and fibrous hyperplasia by HE staining; markedly increased collagen deposition by Sirius red staining (P<0.0001); and elevated protein expression of α-SMA (P<0.01) and FN (P<0.0001). The number of CD31 vascular endothelial cells was increased (P<0.0001) with evident formation of tubular structures, and the count of α-SMA myofibroblasts was also elevated (P<0.0001). In addition, α-SMA myofibroblasts were spatially adjacent to CD31⁺ endothelial cells. ② In vitro experiment results: Compared with the CM-PBS group, the expression levels of α-SMA and FN were significantly increased in myofibroblasts of the CM-DEX group (both P<0.01), while no significant difference was observed in Col Ⅰexpression (P=0.1310). ③ Molecular sequencing: RNA-seq results revealed that 620 genes were upregulated in DEX-treated HUVECs, and 30 of them were identified as pro-fibrotic/inflammation-related. The top 5 genes were PTPRN2, TSC22D3, FKBP5, MAFB and SYP. GO enrichment analysis indicated that these genes were mainly involved in protein binding, transcriptional regulation and protein transport. KEGG enrichment analysis revealed enrichment in TGF-β, JAK-STAT, MAPK, SNARE vesicular transport and exosome pathways. Proteomic analysis identified 20 pro-fibrotic proteins secreted by DEX-treated HUVEC, including TGFBR1, SMAD2, RAB11B and DCN. GO terms were predominantly enriched in extracellular matrix (ECM) and collagen-related functions. KEGG pathways were mainly enriched in the complement and coagulation cascades, ECM-receptor interaction and focal adhesion pathways.

Glucocorticoids induce abnormal activation of vascular endothelial cells, which promotes high expression of pro-fibrotic factors such as DCN, RAB11B, and TGFBR1 through the TGF-β, PI3K-Akt, and vesicle transport/exosome pathways. These factors act on fibroblasts in a paracrine fashion, promote fibroblast activation and excessive synthesis of ECM components including Col Ⅰ and FN. In the reactive zone of the femoral head in GONFH patients, the increased number of vascular endothelial cells and their spatial proximity to myofibroblasts further exacerbate the fibrosis process.