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Background <p>Endothelial-to-mesenchymal transition (EndoMT) contributes to the pathogenesis and rupture of brain arteriovenous malformations (bAVMs), yet its upstream regulatory mechanisms remain largely unclear.</p> Methods <p>S1PR1 expression was first examined across multiple public databases, including the Human Protein Atlas, and subsequently validated in human bAVM tissues via immunohistochemistry and immunofluorescence. Human umbilical vein endothelial cells (HUVECs) with S1PR1 knockdown or overexpression were used to perform transcriptomic (RNA-seq) and proteomic analyses. Enrichment analyses were conducted using Gene Ontology (GO), KEGG, and Reactome databases. EndoMT and cell motility were evaluated by marker expression profiling, Transwell migration, and scratch wound healing assays. In vivo validation was performed using zebrafish models with targeted modulation of <i>s1pr1</i> and <i>ccn1</i> expression.</p> Results <p>S1PR1 expression was markedly reduced in endothelial cells of ruptured human bAVM specimens, as confirmed by immunohistochemistry and dual immunofluorescence. In HUVECs, lentiviral S1PR1 knockdown induced an EndoMT-like phenotype with elevated vimentin, α-SMA, and fibronectin, reduced VE-cadherin and CD31, and enhanced migration. Conversely, S1PR1 overexpression suppressed cell motility. Transcriptomic profiling revealed enrichment of extracellular matrix (ECM) remodeling and basement membrane disassembly pathways, although proteomic analysis confirmed cytoskeletal and junctional disruption. Integrated multi-omics identified CCN1 (CYR61) as a consistently upregulated ECM effector downstream of S1PR1 loss. Zebrafish with <i>s1pr1</i> knockdown or <i>ccn1</i> overexpression exhibited increased cranial hemorrhage, vascular hyperplasia, and erythrocyte extravasation, supporting the in vivo relevance of the S1PR1–CCN1 axis in maintaining cerebrovascular integrity. Silencing CCN1 reversed EndoMT markers and impaired migration in S1PR1-deficient cells, supporting the pathological relevance of the S1PR1–CCN1 axis.</p> Conclusion <p>Our study identifies the S1PR1–CCN1 axis as a critical regulator of endothelial plasticity in bAVMs. S1PR1 knockdown promotes an EndoMT-like phenotype and enhances endothelial migration, although CCN1 acts as a downstream effector mediating these changes. The in vivo findings in zebrafish further highlight the role of this axis in cerebrovascular instability. Targeting this pathway may offer new therapeutic opportunities to mitigate bAVM progression and rupture risk.</p>

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The S1PR1–CCN1 axis drives endothelial-to-mesenchymal transition and vascular instability in brain arteriovenous malformations

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月25日

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  • Ganglei Li,
  • Hongfei Zhang,
  • Mingjian Liu,
  • Yingjun Liu,
  • Shiyu Shen,
  • Sichen Li,
  • Wei Zhu

摘要

Background

Endothelial-to-mesenchymal transition (EndoMT) contributes to the pathogenesis and rupture of brain arteriovenous malformations (bAVMs), yet its upstream regulatory mechanisms remain largely unclear.

Methods

S1PR1 expression was first examined across multiple public databases, including the Human Protein Atlas, and subsequently validated in human bAVM tissues via immunohistochemistry and immunofluorescence. Human umbilical vein endothelial cells (HUVECs) with S1PR1 knockdown or overexpression were used to perform transcriptomic (RNA-seq) and proteomic analyses. Enrichment analyses were conducted using Gene Ontology (GO), KEGG, and Reactome databases. EndoMT and cell motility were evaluated by marker expression profiling, Transwell migration, and scratch wound healing assays. In vivo validation was performed using zebrafish models with targeted modulation of s1pr1 and ccn1 expression.

Results

S1PR1 expression was markedly reduced in endothelial cells of ruptured human bAVM specimens, as confirmed by immunohistochemistry and dual immunofluorescence. In HUVECs, lentiviral S1PR1 knockdown induced an EndoMT-like phenotype with elevated vimentin, α-SMA, and fibronectin, reduced VE-cadherin and CD31, and enhanced migration. Conversely, S1PR1 overexpression suppressed cell motility. Transcriptomic profiling revealed enrichment of extracellular matrix (ECM) remodeling and basement membrane disassembly pathways, although proteomic analysis confirmed cytoskeletal and junctional disruption. Integrated multi-omics identified CCN1 (CYR61) as a consistently upregulated ECM effector downstream of S1PR1 loss. Zebrafish with s1pr1 knockdown or ccn1 overexpression exhibited increased cranial hemorrhage, vascular hyperplasia, and erythrocyte extravasation, supporting the in vivo relevance of the S1PR1–CCN1 axis in maintaining cerebrovascular integrity. Silencing CCN1 reversed EndoMT markers and impaired migration in S1PR1-deficient cells, supporting the pathological relevance of the S1PR1–CCN1 axis.

Conclusion

Our study identifies the S1PR1–CCN1 axis as a critical regulator of endothelial plasticity in bAVMs. S1PR1 knockdown promotes an EndoMT-like phenotype and enhances endothelial migration, although CCN1 acts as a downstream effector mediating these changes. The in vivo findings in zebrafish further highlight the role of this axis in cerebrovascular instability. Targeting this pathway may offer new therapeutic opportunities to mitigate bAVM progression and rupture risk.