Exosome-mediated effects of BRCA1 on cardiovascular artery disease

Figures

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  Fig. 1. Cell Clustering and Annotation from scRNA-seq Data. Note: (A) The foundational framework for studying cell heterogeneity and the progression of CAD atherosclerosis; (B) Analysis based on GSM7445264, GSM7445269, and GSM7445271 datasets downloaded from the GEO database, utilizing UMAP algorithm for dimensionality reduction and clustering visualization, showcasing cell populations isolated from carotid artery samples of CAD atherosclerosis patients, with each color representing a unique cell cluster; (C) Visualization of cell annotations based on UMAP clustering, where each color indicates a cell population; (D) Network analysis of cell communication based on the R package "CellChat"; (E) Analysis of macrophage communication networks with other cells using the R package "CellChat."

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  Fig. 2. The Close Association Between M1 Macrophages and the Progression of CAD. Note: (A) Trajectory skeleton map, mapping cells to pseudotime ordering based on Seurat's cluster ID; (B) Pseudotime analysis showing expression of M1 macrophage markers CD86 and CD80; (C) IHC analysis of CD86 expression in cardiac tissues of Control and ApoE−/− groups; (D) IHC analysis of CD86 expression in carotid tissues of Control and ApoE−/− groups; (E) Flow cytometry analysis of M1 macrophages (CD80+CD86+) in peripheral blood of Control and ApoE−/− groups; (F) Flow cytometry analysis of M2 macrophages (F4/80+CD206+) in peripheral blood of Control and ApoE−/− groups. ***P < 0.001, **P < 0.01, animal experiment n = 6

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  Fig. 3. Communication Analysis Between M1 Macrophages and Cardiomyocytes. Note: (A) Schematic of macrophage and HL-1 cell co-culture model; (B) Flow cytometry detection of HL-1 cell apoptosis post-co-culture, with FLICA+PI+ cells in quadrant Q2 representing apoptotic cells; (C) Immunofluorescence staining to detect apoptosis in HL-1 cells post-co-culture, with white arrows indicating apoptotic cells, Scale bar = 50 μm; (D) Western blot analysis of GSDMD, Caspase-1, and Caspase-4 expression in various cell groups post-co-culture. ***P < 0.001, cell experiment repeated 3 times

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  Fig. 4. Transcriptomic Analysis Combined with WGCNA Algorithm to Identify Key Genes Influencing CAD Progression. Note: (A) Volcano plot of differentially expressed genes from transcriptomic data of normal and model group heart macrophages based on GEO database analysis, including 3 normal and 3 model samples, with red indicating significantly upregulated genes, blue indicating significantly downregulated genes, and grey representing genes with no significant difference; (B) Scale independence, mean connectivity, and scale-free topology plot, determining the weighted value β = 19 to satisfy scale-free network laws; (C) Cluster dendrogram and trait heatmap of transcriptomic data from normal and model group mice samples; (D) Sample selection clustering dendrogram; (E–F) Heatmap (E) and bar graph (F) of gene module correlation with CAD and correlation analysis; (G) Intersection of "pyroptosis" related genes downloaded from GeneCards database with macrophage DEGs and WGCNA selected MEgreenyellow gene module in CAD progression; (H) Expression of intersected genes Brca1, Cdk1, Melk, Rbbp7 in heart macrophages of normal and model group mice. ***P < 0.001, **P < 0.01, *P < 0.05

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  Fig. 5. Regulation of GSDMD and Caspase-1 Expression by BRCA1-Modified Exo. Note: (A) NanoSight particle tracking analysis of nanoscale particle size distribution in Exo derived from macrophages; (B) Transmission electron microscopy analysis of the nanoparticle morphology of Exo from macrophages, left image Scale bar = 500 nm, right image Scale bar = 100 nm; (C) Flow cytometry detection of exosomal markers (CD81 and CD63) expression, with GM130 serving as a negative control; (D) Schematic of HL-1 cells treated with Exo from M1 macrophages; (E) HL-1 cells stained with CellTracker™ Deep Red dye, immunofluorescence staining detecting the interaction between HL-1 cells and Exo, magenta represents HL-1 cells, green represents Exo, white arrows indicate the interaction in merged images, scale bar = 25 μm; (F) CCK8 assay to assess HL-1 cell viability; (G) Flow cytometry analysis of apoptosis in HL-1 cell groups; (H) ELISA measurement of inflammatory markers IL-18 and IL-1β in the supernatant of HL-1 cells treated with Exo; (I) Western blot analysis of GSDMD, Caspase-1, and Caspase-4 expression in HL-1 cells post-exosome treatment. ***P < 0.001, **P < 0.01, *P < 0.05, cell experiment repeated 3…

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  Fig. 6. Exo from Macrophages Delivering BRCA1 Influence SMC Cell Function. Note: (A) Immunofluorescence analysis of the distribution in the heart, liver, spleen, and lungs after exosome injection in an animal model, scale bar = 25 μm; (B) Immunofluorescence analysis of cell types with exosome distribution, scale bar = 25 μm; (C) H&E staining of carotid artery tissues from various mouse groups, scale bar = 200 μm; (D-E) IHC staining for αSMA expression, a smooth muscle cell marker, in carotid artery tissues of mouse groups, Scale bar = 100 μm; (F-G) Immunofluorescence staining to assess smooth muscle cell proliferation in carotid artery tissues, αSMA indicates smooth muscle cells, Ki67 indicates proliferating cells, DAPI indicates nuclei, white arrows point to αSMA + Ki67 + double-positive cells, scale bar = 25 μm. ***P < 0.001, **P < 0.01, *P < 0.05, ns P > 0.05, animal experiment n = 6

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  Fig. 7. Mechanistic Study of BRCA1-Modified Exo in Coronary Artery Atherosclerosis. Note: (A) Schematic of atherosclerosis model in 8-week-old ApoE−/− mice fed a high-fat diet for 12 weeks and treated with BRCA1-modified Exo; (B) Representative images of atherosclerotic plaques (yellow arrows) in the aorta and its branches of mouse groups; (C) Representative images and quantification of Oil red O staining in the entire aorta lesion area of mouse groups; (D) Representative images and cross-sectional plaque area statistics of aortic root H&E staining in mouse groups, Scale bar = 200 μm; (E) Representative images and analysis of Oil red O staining in the aortic root of mouse groups, Scale bar = 200 μm; (F) IHC staining for GSMDM expression in cardiac tissues of mouse groups, Scale bar = 50 μm; (G) Flow cytometry analysis of the proportion of pyroptosis in cardiomyocytes of model, negative control, and BRCA1-silenced groups, with flow cytometry results and statistical bar graphs displayed; (H) Western blot analysis of the expression of GSDMD, Caspase-1, and Caspase-4 in the heart tissues of each group of mice; (I) qRT-PCR analysis of BRCA1 mRNA expression levels in M1 macrophages before and…