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.

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.

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.

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.

After ischemic stroke, neutrophils hyperactivate, increasing in number and worsening inflammation, causing neural damage. Prior scRNA-seq showed Lrg1 modulates cells subsentence to cerebral ischemia-reperfusion injury, but its mechanism in regulating neutrophil accumulation/differentiation post-injury is unclear.

Lrg1 knockout impact on neutrophil accumulation was assessed via immunofluorescence and western blot. Three-dimensional reconstruction of immunofluorescent staining analyzed cell-cell interactions among neutrophils and microglia. scRNA-seq of WT and Lrg1^-/- mice from GSE245386 and GSE279462 was conducted. Each group conducted oxidative phosphorylation scoring via Gene Set Enrichment Analysis (GSEA), while Metascape was employed to perform GO and KEGG enrichment analyses for elucidating functional mechanisms. CellChat exhibited cell-cell communication. Furthermore, alterations in microglial phagocytic activity were evaluated by immunostaining for CD68, a well-established marker of phagolysosomal activity in phagocytic cells. Brain energy metabolism was evaluated via glutamate dehydrogenase activity and ATP levels with ELISA, and enzyme expression was analyzed by immunofluorescence and western blot.

Lrg1 knockout decreased neutrophil accumulation and NET formation in mice. 3D immunofluorescence reconstruction confirmed neutrophil co-localization with endothelial cells/microglia. scRNA-seq revealed that the oxidative phosphorylation score was significantly higher in the MCAO/R+WT group compared to both the Sham-operated+WT and Lrg1^-/- groups. Notably, the oxidative phosphorylation score was further elevated in the MCAO/R+Lrg1^-/- group. Immunostaining showed that Lrg1 knockout elevated CD68+ lysosome expression post-MCAO/R, with TMEM119 colocalizing with these lysosomes. MCAO/R raised CD68 expression in ischemic brains, an effect further intensified by Lrg1 knockout. KEGG analysis linked differential genes to oxidative phosphorylation pathways. Validation in MCAO/R vs. sham groups revealed increased ROS production and reduced expression of complex enzymes I-V (NDUFB8, SDHB, UQCRC1, MTCO2, ATP5A1). Lrg1 intervention increased enzyme expression. Immunofluorescence and western blot in brain tissue showed similar patterns in microglia and enzymes I-V.

Lrg1 knockout significantly enhances microglial phagocytic activity towards neutrophils subsequent to cerebral ischemia-reperfusion injury, through its regulatory effect on the oxidative phosphorylation pathway. This finding accentuates Lrg1 as a highly potential therapeutic target for intervening in and modulating post-ischemic inflammatory responses.