Integrative Analysis of scRNA-Seq and Bulk RNA-Seq Reveals Novel Transcription Factor Regulating Endothelial Heterogeneity Induced by Lrg1 Following Cerebral Ischemia-Reperfusion
SHAOFENG XIONG1,2, WENKAI LV3, GUOSHENG CAO4, LONGSHENG FU1, WEN LIU3, MENGFAN LEI2, YANNI LV1,5,*
1 Department of pharmacy, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, 330006, China
2 School of Pharmacy, Jiangxi Medical College, Nanchang University, Nanchang, 330036, China
3 Institute of Chinese Materia Medica, Jiangxi Provincial Institute of Traditional Chinese Medicine, Nanchang, 330077, China
4 College of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430000, China
5 Key Laboratory of Rare Neurological Diseases of Jiangxi Provincial Health Commission, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, 330036, China
* Corresponding Author: YANNI LV. Email: 
BIOCELL https://doi.org/10.32604/biocell.2025.073401
Received 17 September 2025; Accepted 26 November 2025; Published online 22 December 2025
Abstract
Objective: Leucine-rich alpha-2 glycoprotein 1 (Lrg1) could regulate diverse cells in cerebral ischemia-reperfusion. Our study seeks to uncover Lrg1’s impact on endothelial cell heterogeneity via differentiation pathways and transcription factors.
Method: The CSOmap model measured cell-to-brain-center distances using single-cell RNA sequencing (scRNA-seq) data in middle cerebral artery occlusion reperfusion (MCAO/R). Monocle2 mapped endothelial differentiation paths. Gene set enrichment analysis (GSEA) analyzed endothelial subcluster variations. Database searches revealed a zinc finger MIZ-type containing 1 protein-frizzled 3 (Zmiz1-Fzd3) promoter interaction. Endothelial cells were transfected with a Fzd3 promoter-luciferase plasmid. Polymerase chain reaction (PCR) and western blotting assessed MCAO/R or Zmiz1 overexpression effects on Fzd3-related mRNA and proteins. A retroviral vector carrying Zmiz1 was injected into the brains of mice to study its effect on Fzd3.
Result: Lrg1−/− mice exhibited elevated cell adhesion proteins and decreased microvascular leakage after MCAO/R. CSOmap showed widened astrocyte spacing in these mice. RSS revealed Zmiz1 overexpression in MCAO/R+
Lrg1−/− mice. MCAO/R and pcDNA3-Zmiz1 transfection both enhanced luciferase activity with Fzd3, indicating Zmiz1 binding to Fzd3. Retroviral Zmiz1 injection or knockdown disrupted ischemic brain tight junctions, highlighting Zmiz1’s key role in blood-brain barrier protection, likely through Fzd3 pathway modulation.
Conclusion: The findings indicate
Lrg1 knockout induces endothelial differentiation by activating Zmiz1, which is crucial for maintaining blood-brain barrier function, possibly via modulating the Fzd3 pathway.
Keywords
Single-cell RNA sequencing; endothelial heterogeneity; leucine-rich alpha-2 glycoprotein 1; zinc finger MIZ-type containing 1 Protein; Frizzled 3; transcription factor; cerebral ischemia reperfusion
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