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Discovery, prioritization, validation, and replication of perturbed GVU vascular target-astrocyte ligand pair SMAD3-VEGFA. a Strength and direction of NicheNet vascular target-astrocyte ligand interactions. Left: predicted ligands in astrocyte clusters. Right: predicted targets in vascular clusters. Edge: regulation strength between ligands and target genes; Cyan astrocyte, purple: endothelial markers. Direction of change in AD is denoted as blue for up and red for downregulation. b Of the perturbed vascular targets in AD brains, SMAD3, which is upregulated in AD pericytes and has strong astrocytic connections, is prioritized. Of the astrocytic ligands, VEGFA, which is downregulated in AD and has strong predicted interactions with SMAD3, is prioritized. c, d We validated expression of SMAD3 in vascular cells and VEGFA expression in astrocyte cells through RNAscope (scale bar:100 µm) and immunofixation (scale bar:10 µm). e Immunohistochemistry results showed significantly higher phospho-SMAD3 immunoreactivity in AD compared to controls in pericytes (p < 0.01, n = 10 per diagnosis). fSMAD3 and VEGFA brain expression changes in external brain snRNAseq studies. Pericytes (purple) and astrocytes (cyan) were from multiple studies and were clustered (Gray dots: other nuclei). In forest plots, the square indicates the coefficient, which is the natural log(fold change). The left bar: 2.5% confidence interval; the right bar: 97.5% confidence interval. (Ast: astrocytes, Per: pericytes, TCX: temporal cortex, MTX: midtemporal cortex, EC: entorhinal cortex, DLPFC: dorsolateral prefrontal cortex, PFC: prefrontal cortex, SFX: superior frontal cortex, Hippocampus: HC, AG: angular gyrus, TH: thalamus) g 6 intronic variants associated with higher blood expression levels of SMAD3 (eQTL) were also associated with decreased brain infarcts in ADNI, MCSA, and meta-analyzed cohorts. P-values and direction of effects from the infarct GWAS and the eQTL analysis in MCSA, ADNI, and meta-analysis (random effects) are shown. h, i Whole-brain association analysis of blood SMAD3 levels with brain Aβ deposition and cortical thickness in the ADNI cohort. Color scales indicate regions where higher blood SMAD3 were associated with less brain amyloid-β deposition and less brain atrophy, respectively. Statistical maps were thresholded for a multiple testing adjustment to a corrected significance level of 0.05. Source data are provided as a Source Data file.
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