Targeting angiopoietin like-2 positive senescent cells improves cognitive impairment in adult male but not female atherosclerotic LDLr−/−;hApoB100+/+ mice

Figures

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  Fig. 1. Impact of sh-angptl2 treatment on cognition in the MWM test. A Latency to find the hidden platform from day 1 to day 5 in the acquisition phase (learning memory) of the MWM test in male (A–B) and female (C–D) ATX mice after treatment with sh-SCR or sh-angptl2. Each value is a mean of four trials performed by each mouse, per day of learning. ‡: p < 0.05 sh-SCR vs. sh-angptl2, in each day of learning, two-way ANOVA with repeated measures (Days × Treatment). Latency to target was also analyzed to determine in each group at what day the mice started to learn the localization of the hidden platform in male (B) and female (D) mice: * p < 0.05 vs. Day 1, two-way ANOVA with repeated measures (Days × Treatment) and Dunnet’s multiple comparisons test. (E, F, G, H) Percentage of time spent in the target quadrant (TQ, quadrant in which the platform was hidden) and in the opposite quadrant (OQ) by male and female mice after treatment with sh-SCR or sh-angptl2 during the probe test. This probe test was performed on day 5, 1 h after the last acquisition of the learning phase (short-term memory retention, E–F) and 72 h after the end of the learning phase (delayed memory retention, G–H). *: p <…

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  Fig. 2. Impact of sh-angptl2 treatment on atherosclerotic lesion, blood flow index, carotid compliance and cerebral endothelial dilatory function. A Typical images of thoracic aortas with atheroma plaques; lesions are expressed as percentage of total aortic area in both male and female ATX mice treated with sh-SCR or sh-angptl2 (n = 12 males, n = 10–13 females). *: p < 0.05 vs. male-sh-SCR, two-way ANOVA (Treatment × Sex) and Tukey’s multiple comparisons test. B Time-series fluorescence imaging of ICG on a mouse head scalp and calculation of the cerebral BFI. A typical image of the fluorescent signal measured trough the open scalp of a mouse is presented. Six regions of interest (primary visual cortex (V1), retrosplenial cortex (RS) and primary motor cortex (M1), left (L) and right (R) brain) were scanned for fluorescence acquisition. Fluorescence was averaged within the six regions; the slope of the averaged time-series fluorescence signal corresponds to BFI. Two-way ANOVA (Treatment × Sex) and Tukey’s multiple comparisons test. C Carotid compliance of both male and female mice treated with sh-SCR or sh-angptl2 (n = 8 males, n = 7–9 females), expressed as circumferential strain (%) in…

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  Fig. 3. Impact of sh-angptl2 treatment on angptl2 protein and gene expression. Typical images of confocal immunostaining for angptl2 (in red; nuclei stained in blue with DAPI) in hippocampal slices from male (A) and female (B) ATX mice treated with sh-SCR or sh-angptl2. All images were taken in the dentate gyrus of the hippocampus. C Average hippocampal expression of angptl2 in male and female ATX mice treated with sh-SCR or sh-angptl2 analyzed using Image J. Two-way ANOVA (Sex × Treatment) and Tukey’s multiple comparisons test. Data are mean ± SEM of n = 18 images collected in three mice, per group. D Angptl2 gene expression measured by RT-qPCR in native aortic EC scraped from the aorta the day of the sacrifice, in mice after 1, 2 or 4 weeks of treatment with sh-angptl2. *: p < 0.05: vs. untreated mice on week 0, one-way ANOVA and Dunnett’s multiple comparisons test. Data are expressed as mean ± SEM of n = 6 mice of both sexes. E Angptl2 gene expression measured by RT-qPCR in the hippocampal region in mice after 1, 2 or 4 weeks of treatment with sh-angptl2. Data are expressed as mean ± SEM of n = 6 mice of both sexes

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  Fig. 4. Impact of sex and sh-angptl2 treatment on brain cell types from ATX mice. A Uniform manifold approximation and projection plot, in two dimensions (UMAP-1 and UMAP-2), of annotated cell-types clustered based on their specific gene expression, present in hippocampus from male and female ATX mice treated with sh-SCR (A) or sh-angptl2 (B). C Dot plot showing the expression of the top 10 gene markers used to identify seven distinct cell clusters. In the plot, dot color represents average gene expression, with values scaled from -1.0 (blue) to 2.0 (red), dot size reflects the percentage of cells expressing each gene, ranging from 0% (smallest) to 75% (largest). D–E Feature UMAP plots showing the distribution of angptl2 expression across the entire data set, stratified by sex (male or female mice) and treatment (sh-SCR in D or sh-angptl2 in E); average expression of angptl2 is indicated by color with values scaled from -1.0 (blue) to 2.0 (red)

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  Fig. 5. Impact of sex and sh-angptl2 treatment on senescent pathways. Dotplot of senescent (A) and apoptotic (B) markers gene expression in hippocampal cell types across the four different conditions. Average expression of senescent and apoptotic markers is indicated by color with values scaled from -1.0 (blue) to 2.0 (red); the percentage of cells expressing the markers is indicated by dot size, scaling from 0 to 30% (for senescence) or from 20 to 60% (for apoptosis). C–F Heatmaps displaying GSVA scores for selected senescence-related gene sets across hippocampal cell types; comparisons are shown between male and female sh-SCR (C), male and female sh-angptl2 (D), between male sh-SCR and male sh-angptl2 (E) and between female sh-SCR and female sh-angptl2 (F). Rows represent senescence gene-related pathways (log2 fold change expression), columns represent cell types. The color of each cell in the matrix represents the expression level of a specific pathway: red corresponds to higher expression, blue corresponds to lower expression, in male vs. female-sh-SCR, scaling from -0.2 to 0.2 (C), in male vs. female-sh-angptl2, scaling from -0.4 to 0.4 (D), in male-sh-angptl2 vs. male-sh-SCR,…

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  Fig. 6. Differentially expressed genes in the three main cell types involved in the sex-specific response to sh-angptl2 treatment in mice. Identification of the cell types most responsive to biological perturbations in the data set using Augur method: identification of cell-specific transcriptomic effect of the treatment (sh-angptl2 and sh-SCR) (A) and of the sex (B) in hippocampal cell types. Augur provides an unbiased AUC score, which measures how well the model can separate two groups of cells based on their gene expression profiles: the higher the AUC (> 0.5 and the closest to 1.0) in a cell type, the most likely this cell type is involved in the biological response. C, D, E Venn diagrams showing the overlap of DEGs, both upregulated and downregulated, in male and female cells following sh-angptl2 treatment. Associated gene pathways (Gene Ontology Biological Process; p < 0.05, log₂ fold change ≥ 0.25, minimum percent expression ≥ 10%) were identified using EnrichR for neurons (C), CP-epithelial cells (D) and OPCs (E). In the associated gene pathway analysis, bar length corresponds to the number of DEGs associated with each pathway, reflecting the strength of the gene set enrichment

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  Fig. 7. Cell–cell communication between neurons, OPCs and CP-epithelial cells in the brain of male and female ATX mice treated with sh-SCR or sh-angptl2. A Cell–cell communication analysis using the Connectome package, between OPCs, neuronal and CP-epithelial cells from male and female hippocampus, comparing the transcriptomic impact of sh-angptl2 vs sh-SCR on ligands (left panels), receptors (middle panels) and ligand-receptors (right panels) perturbations. B Circos plots of most impacted ligand-receptor pairs by sh-angptl2 in male and female neuronal-OPCs-CP-epithelial cells, with connecting lines representing significant communication links in the signaling network. The stringent significance criteria (log₂ fold change ≥ 0.25, gene expression present in at least 10% of cells per cell type, and p value < 0.05) were applied, and restricted to the three cell types identified by Augur