Journal: Advanced Science

Article Title: Lilrb4a Suppression Reprograms Microglia to Mitigate APOE4‐Associated Amyloid Plaques and Cerebral Amyloid Angiopathy in Association With a PPAR‐Linked Pro‐Clearance State

doi: 10.1002/advs.202524167

Figure Lengend Snippet: LILRB4 shows stronger APOE4‐associated Co‐IP signals in the tested contexts and is upregulated in APOE4 and amyloid‐related settings. (A,B) Reciprocal Co‐IP of human APOE4 with Flag‐tagged human LILRB3 (A) or Flag‐tagged human LILRB4 (B) in HEK293T cells. Schematic representation of Co‐IP between LILRB3/LILRB4 and APOE4 protein. Immunoprecipitation with anti‐APOE or anti‐Flag antibodies, followed by immunoblotting for APOE4 and LILRB3/LILRB4. Input lysates are shown as positive controls, and IgG immunoprecipitation serves as a negative control; (C, D) Reciprocal Co‐IP of endogenous APOE and LILRB4 in microglia isolated from 12‐month‐old APOE4 (C) or APOE3 (D) mice 24 h after intraperitoneal LPS injection. Cell lysates were immunoprecipitated with anti‐APOE or anti‐LILRB4 antibodies and immunoblotted as indicated. Input lysates are shown as positive controls, and IgG immunoprecipitation serves as a negative control; (E) Representative Lilrb4a immunostaining in cortical sections from LPS‐treated APOE3 and APOE4 mice (10×, scale bar = 500 µm); (F) Quantification of Lilrb4a‐positive area in the cortex ( n = 4–5); (G) Schematic diagram of the experimental design; (H) Representative confocal images of X‐34 (blue), APOE (red), and IBA1 (green) staining in cortical sections from 5EL and 5ELKO mice; arrows indicate plaque‐associated microglia containing APOE signal (60× oil, scale bar = 50 µm); (I) Quantification of APOE‐positive area within IBA1‐positive microglia surrounding plaques (APOE/IBA1 area) ( n = 9–12); (J, K) Lilrb4a mRNA expression was analyzed by qPCR in cortical tissues from C57 and 5xFAD mice of different ages ( n = 5–8); Unless otherwise specified, experiments not shown as sex‐separated in the figures were performed using male mice only (Applicable to all figure legends). Data are presented as mean ± standard error of the mean (SEM). Each dot represents one mouse. Statistical significance was determined by unpaired two‐tailed Student's t test (F), one‐way ANOVA (J,K), or two‐way ANOVA (I), as appropriate. * p <0.05, ** p <0.01.

Article Snippet: For phagocytosis assays in sorted primary microglia from APOE3 and APOE4 mice, cells were treated with ASO‐L4a or control ASO (5 pmol per 1 × 10 4 cells) for 36 h, followed by an additional 24 h in medium containing either a PPAR‐γ agonist (MCE, HY‐13956, 20 μ m ) or a PPAR‐γ inhibitor (MCE, GW9662, 10 μ m ).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Negative Control, Isolation, Injection, Immunostaining, Staining, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Lilrb4a Suppression Reprograms Microglia to Mitigate APOE4‐Associated Amyloid Plaques and Cerebral Amyloid Angiopathy in Association With a PPAR‐Linked Pro‐Clearance State

doi: 10.1002/advs.202524167

Figure Lengend Snippet: Exploratory bulk RNA‐seq nominates a PPAR‐related signature, and PPAR‑γ activation phenocopies clearance‐associated outcomes. (A) Volcano plot of bulk RNA‐seq data from thalamic tissue of 5EL and 5ELKO mice; colored points indicate genes meeting an exploratory threshold of nominal p <0.05 and absolute fold change >1.2; (B) KEGG pathway enrichment analysis of transcriptomic alterations in 5ELKO versus 5EL mice; (C) qPCR validation of selected genes related to the PPAR signaling pathway, including Apoa1 , Fabp7 , Pck1 , Plin2 , Apoa2 , Pparg , Cyp2e1 in thalamus from 5EL and 5ELKO mice ( n = 4–5); (D) Representative RNAscope images of Pparg mRNA in brain sections from 5EL and 5ELKO mice, combined with X‐34 and IBA1 co‐staining (left) and Imaris‐based reconstruction (right). White arrows indicate PPAR‐γ‐positive signals within microglia (60× oil, scale bar = 20 µm); (E) Quantification of Pparg RNAscope signal volume within plaque‐associated microglia in 5EL and 5ELKO mice; (F) Representative Incucyte images of pHrodo‐labeled Aβ uptake in BV2 cells treated with vehicle or a PPAR‐γ agonist at 0 h and 1.5 h; (G) Quantification of the phagocytosis rate in BV2 cells under the indicated agonist or inhibitor treatments, expressed as the percentage of pHrodo‐positive area relative to total cell area ( n = 3); (H) qPCR analysis of Lilrb4a and Pparg expression in BV2 cells treated with vehicle or a PPAR‐γ agonist ( n = 3); (I) Schematic diagram of the FAM‐labeled Aβ phagocytosis and degradation assays in BV2 cells; (J) Representative flow cytometry histograms showing intracellular Alexa‐488 fluorescence in BV2 cells after 1.5 h phagocytosis and after 3 h loading followed by 24 h degradation under the indicated PPAR‐γ agonist or inhibitor conditions; (K) Quantification of mean intracellular Alexa‐488 fluorescence in BV2 cells after 1.5 h phagocytosis (left) and after 24 h degradation (right) ( n = 3); (L) Representative flow cytometry histograms showing intracellular Alexa‐488 fluorescence in sorted primary microglia from LPS‐treated APOE4 mice under the indicated ASO and PPAR‐γ inhibitor conditions. Left, phagocytosis assay, in which cells were incubated with FAM‐Aβ and analyzed after 1.5 h to assess uptake. Right, degradation assay, in which cells were first allowed to internalize FAM‐Aβ for 3 h, then switched to substrate‐free medium, and analyzed 24 h later to assess residual intracellular signal; (M) Quantification of mean intracellular Alexa‐488 fluorescence in APOE4 microglia in the phagocytosis (left) and degradation (right) assays ( n = 3); (N,O) Corresponding representative histograms and quantification for sorted primary microglia from LPS‐treated APOE3 mice under identical assay conditions and treatment paradigms, with phagocytosis measured at 1.5 h and degradation measured 24 h after 3 h loading with FAM‐Aβ ( n = 3); (P) Representative immunofluorescence images of WT primary neuron–microglia co‐cultures treated with vehicle‐ or PPAR‐γ agonist‐treated microglia, showing DAPI (blue), MAP2 (green), and IBA1 (red) (10×, scale bar = 200 µm); (Q) Quantification of neuronal survival in co‐culture, measured as MAP2‐positive area ( n = 8–11). Data are presented as mean ± SEM. Statistical significance was determined by unpaired two‐tailed Student's t test (C, E, H, and Q) or one‐way ANOVA (G, K, M, and O), as appropriate. ns, not significant. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: For phagocytosis assays in sorted primary microglia from APOE3 and APOE4 mice, cells were treated with ASO‐L4a or control ASO (5 pmol per 1 × 10 4 cells) for 36 h, followed by an additional 24 h in medium containing either a PPAR‐γ agonist (MCE, HY‐13956, 20 μ m ) or a PPAR‐γ inhibitor (MCE, GW9662, 10 μ m ).

Techniques: RNA Sequencing, Activation Assay, Biomarker Discovery, RNAscope, Staining, Labeling, Expressing, Flow Cytometry, Fluorescence, Phagocytosis Assay, Incubation, Degradation Assay, Immunofluorescence, Co-Culture Assay, Two Tailed Test

Journal: Bioactive Materials

Article Title: Apolipoprotein E knockout attenuates vascular graft fibrosis by reducing profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1016/j.bioactmat.2026.01.029

Figure Lengend Snippet: Profibrotic macrophages increasing fibroblast proliferation via their secreted IGF-1. (a) Dot plots of Igf1 expression in C2 and the other subgroups of macrophages in regenerated aortas 30 and 90 days after graft implantation in WT and Apoe −/− rats. (b) Igf1 expression score in macrophages in regenerated aortas 30 and 90 days after graft implantation in WT and Apoe −/− rats. ∗∗∗∗ indicates p < 0.0001, unpaired t -test. (c) Quantification of IGF-1 concentration in regenerated aortas 30 and 90 days after graft implantation in WT and Apoe −/− rats. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each time point and each group, five different samples from five different animals were analyzed (n = 5). UMAP of fibroblasts in regenerated aortas 30 days (d) and 90 days (e) after graft implantation in WT and Apoe −/− rats, heatmap of cell cycle (Ccnd1, Ccnd2, and Ccnd3) scores in UMAP of fibroblasts, and box plots of cell cycle scores in fibroblasts. ∗∗∗∗ indicates p < 0.0001, unpaired t -test. (f) Immunofluorescence staining of Ki67 in regenerated aortas 30 and 90 days after graft implantation in WT and Apoe −/− rats. L indicates lumens. (g) Quantification of Ki67 positive cells in regenerated aortas. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each time point and each group, five different images from five different animals were analyzed (n = 5). (h) Quantification of IGF-1 in culture mediums of WT and APOE KO macrophages after their culture on PCL scaffolds for 48 h by ELISA. ∗∗ indicates p < 0.01, unpaired t -test. For each group, three different samples were analyzed (n = 3). (i) Immunofluorescence staining of Ki67 in WT and APOE KO fibroblasts after treatment with IGF-1 (10 ng/mL) for 24 h. Cells were counterstained with phalloidin. (j) Quantification of proliferation of WT and APOE KO fibroblasts treated with IGF-1 (10 ng/mL) for 24 h using cell counting kit-8 (CCK-8). ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, three different samples were analyzed (n = 3). (k) Quantification of proliferation of WT fibroblasts treated with conditioned medium (CM) with or without IGF-1 blocking antibody (Ab, 1 μg/mL) for 24 h using CCK-8. CM were medium conditioned by WT macrophages cultured on PCL scaffolds for 48 h ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, three different samples were analyzed (n = 3).

Article Snippet: Exogenous APOE (0.25 μg/mL, MCE, HY-P701096), TGF-β1 (10 ng/mL, MCE, HY-P7117), IGF-1 (10 ng/mL, MCE), conditioned medium by macrophages, or IGF-1 blocking antibody (1 μg/mL, Invitrogen, MA5-18035) was added into culture medium and incubated with WT or APOE KO fibroblasts for 24 h.

Techniques: Expressing, Concentration Assay, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Cell Counting, CCK-8 Assay, Blocking Assay, Cell Culture

Journal: Bioactive Materials

Article Title: Apolipoprotein E knockout attenuates vascular graft fibrosis by reducing profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1016/j.bioactmat.2026.01.029

Figure Lengend Snippet: Downregulation of APOE by AAV ameliorating fibrosis during vascular regeneration after graft implantation in vivo . (a) Illustration of a strategy of adventitial delivery of AAV-shRNA(Apoe) to inhibit APOE levels in regenerated aortas after graft implantation in vivo . Two weeks after graft implantation in vivo , AAV-shRNA(Apoe) were injected into the adventitia of the regenerated aortas, which were then harvested for analysis three weeks later. (b) M mode images of ultrasound detection of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. Arrow heads indicate movement of vascular walls. (c) Tensile tests of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. (d) Quantification of RI, PI, and compliance of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different images from six different animals were analyzed (n = 6). (e) Quantification of elastic modulus of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different images from six different animals were analyzed (n = 6). (f) H&E, MTC and EVG staining of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. (g) Immunofluorescence staining of COL I, COL III, elastin, αSMA, and eNOS in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. L indicates lumens. Arrow heads indicate capillaries. Quantification of adventitia thickness (h), collagen positive areas according to MTC staining (i), elastin positive areas according to EVG staining (j), COL I positive areas (k), COL III positive areas (l), and number of capillaries (m) in adventitial areas of regenerated aortas. (n) Immunofluorescence staining of CTSD and CD68 in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. (o) CD68 and CTSD double positive cells in regenerated aortas. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different samples from six different animals were analyzed (n = 6). (p) WB results of APOE, CTSD and SPP1 levels in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks and quantification of levels of APOE, CTSD and SPP1 in regenerated aortas. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different samples from six different animals were analyzed (n = 6). (q) Quantification of IGF-1 concentrations in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks by ELISA. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different samples from six different animals were analyzed (n = 3).

Article Snippet: Exogenous APOE (0.25 μg/mL, MCE, HY-P701096), TGF-β1 (10 ng/mL, MCE, HY-P7117), IGF-1 (10 ng/mL, MCE), conditioned medium by macrophages, or IGF-1 blocking antibody (1 μg/mL, Invitrogen, MA5-18035) was added into culture medium and incubated with WT or APOE KO fibroblasts for 24 h.

Techniques: In Vivo, shRNA, Injection, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay