Comprehensive Analysis and in Vitro Verification of Endothelial-mesenchymal Transition in Moyamoya Disease

Introduction: Moyamoya disease (MMD) is a rare, chronic, and progressive cerebrovascular disorder, yet its underlying causes and mechanisms remain unclear. Previous studies have suggested a potential involvement of endothelial-mesenchymal transition (EndMT) in the pathogenesis of MMD. This study aimed to explore the contribution of EndMT-related genes (ERGs) in MMD.

Methods: Two datasets, GSE141022 and GSE157628, were intergrated as the training set after batch effects removal. Differentially expressed ERGs were identified between control and MMD groups. Functional enrichment analysis and immune infiltration analysis were further performed. LASSO regression was used for hub MMD-related ERGs selection. Consensus clustering was used for MMD subtype classification based on these hub MMD-related ERGs. Molecular characteristics between MMD subtypes were analyzed by WGCNA. PPI network was used to illuminate the genetic relationship. The hub MMD-related ERGs were validated in an independent testing set, GSE189993. Nomogram models were constructed and evaluated by ROC curves and calibration plots. Additionally, CCK-8, EdU, wound healing, and western blot were performed to confirm the function of the hub MMD-related ERGs.

Results: A total of 107 DE-ERGs were identified. Functional enrichment analysis showed these genes were associated with EndMT and immune response. The infiltrating levels of immune cells were commonly higher in MMD group. LASSO regression identified 12 hub MMD-related ERGs, leading to the identification of two MMD subtypes. Four ERGs emerged as the final hub MMD-related ERGs after validation in the testing set, including CCL21, CEBPA, KRT18, and TNFRSF11A. Nomogram models exhibited excellent discrimination ability. In vitro experiments showed that CCL21, CEBPA, KRT18 and TNFRSF11A could promote proliferation, migration and EndMT.

Conclusion : This study investigated the potential role of EndMT in MMD and identified 4 hub MMD-related ERGs, which provided potential therapeutic targets for MMD treatment.