Open Access

ARTICLE

Integrative Analysis of scRNA-Seq and Bulk RNA-Seq Reveals Novel Transcription Factor Regulating Endothelial Heterogeneity Induced by Lrg1 Following Cerebral Ischemia-Reperfusion

SHAOFENG XIONG^1,2, WENKAI LV³, GUOSHENG CAO⁴, LONGSHENG FU¹, WEN LIU³, MENGFAN LEI², YANNI LV^1,5,*

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 2026, 50(1), 12 https://doi.org/10.32604/biocell.2025.073401

Received 17 September 2025; Accepted 26 November 2025; Issue published 23 January 2026

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.

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Supplementary Material

Supplementary Material File

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