Prevention of atrial fibrillation after open-chest surgery with extracellular vesicle therapy

Figures

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  Figure 1. Characterization of extracellular vesicles produced by human atrial explant-derived cells cultured under serum-free, xenogen-free culture conditions within a clinical cell manufacturing facility. (A) Proteomic array demonstrating presence of markers indicative of EV identity while lacking cellular contaminants. (B) Flow cytometry demonstrating the relationship between EV size and surface marker expression (n = 3 biological replicates). One-way ANOVA with individual-mean comparisons by Bonferroni’s multiple 2-tailed comparisons test. (C) Simple linear regression of the relationship between EV concentration and acetylcholinesterase activity (n = 5 biological replicates). Data are plotted showing the 95% confidence bands of the best fit line. (D) Relative abundance of microRNA transcripts within EDC EVs (n = 3 biological replicates). EV, extracellular vesicles; FDR, false discovery rate; miR, microRNA.

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  Figure 2. Human atrial explant-derived cells cultured under serum-free, xenogen-free culture conditions within a clinical cell manufacturing facility produce extracellular vesicles that contain antifibrotic/antiinflammatory proteins. Relative abundance of proteins within EDC EVs (n = 3 biological replicates). Enrichment map of the EDV EV proteome demonstrating the relationship between biological pathways that are expressed more than would be expected by chance. Pathways are represented by circles (nodes) connected with lines (edges) according to the number of shared proteins. Node size reflects the number of proteins participating in each pathway. The number of proteins shared by connected pathway nodes determines the thickness of edges. Upregulated pathways are colored red while downregulated pathways are in blue. The color of the concentric circles around each node represents the level of enrichment based on the enrichment score scale provided. Groupings of pathways are labeled by common activities or functions. APC, anaphase-promoting complex; ECM, extracellular matrix; LFQ, label-free quantification; MMP2, matrix metalloproteinase 2; NIK, NF-κB–inducing kinase.

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  Figure 3. Human atrial extracellular vesicle effects on postoperative AF and atrial structure. (A) Study schematic demonstrating the study design, numbers of animals included, group allocations, and outcomes measured. One additional female rat was inadvertently randomized to receive extracellular vesicles (EVs) and was included in the final analysis (n = 18 female talc+EV group only). (B) Two representative tracings showing the effect of EV treatment on talc-treated animals after burst pacing. (C) Effect of EV treatment on the probability of inducing AF after burst pacing. Superimposed numbers indicate the absolute number of female (lower gray bar + number) or male (upper black bar + number) animals that went into AF. Logistic regression using a generalized linear model with binomial probability distribution and the logit link function was performed. (D) Effect of EV treatment on the mean duration of AF episodes (n = 2–25). (E) Effect of EV treatment on hydroxyproline content (n = 10), Masson’s trichrome fibrosis (n = 6–8), and weight (n = 10). One-way ANOVA with individual-mean comparisons by Bonferroni’s multiple 2-tailed comparisons test. AF, atrial fibrillation; EPS,…

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  Figure 4. Effect of human atrial extracellular vesicles on inflammation. (A) Representative images of atrial tissue sections after hematoxylin and eosin staining showing the effect of talc and extracellular vesicles (EVs) on inflammatory cell infiltration. Quantitative analysis showing the effect of talc and EVs on inflammatory cell infiltration (n = 8–12 biological replicates). (B) Effect of talc and EVs on interleukin-1β (IL-1β), IL-2, IL-6, IL-18, monocyte chemoattractant protein–1 (MCP-1), platelet-derived growth factor AB (PDGF-AB), transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) expression (n = 4–6 biological replicates). One-way ANOVA with individual-mean comparisons by Bonferroni’s multiple 2-tailed comparisons test. Scale bar, 2 mm or 200 μm, as indicated.

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  Figure 5. Effect of human atrial extracellular vesicles on inflammatory cell infiltration. (A) Effect of explant-derived cell (EDC) extracellular vesicles (EVs) on the number of neutrophils (CD11b+), cytotoxic T cells (CD3+), and T helper cells (CD4+) found within treated atria (n = 5 biological replicates, 6 random fields per biological replicate). (B) Effect of talc and EVs on the atrial content of pro-inflammatory M1 macrophages (CD68+) and antiinflammatory M2 macrophages (CD163+, n = 5 biological replicates, 6 random fields per biological replicate). One-way ANOVA with individual-mean comparisons by Bonferroni’s multiple 2-tailed comparisons test. HPF, high-power field.

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  Figure 6. Effect of human atrial extracellular vesicles on atrial fibroblast proliferation. (A) Effect of explant-derived cell (EDC) extracellular vesicles (EVs) on the relative cell number, population doubling time, and nuclear incorporation of the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) within atrial fibroblasts at baseline and after treatment with interleukin-6 (IL-6), transforming growth factor β1 (TGF-β1), and/or EDC EVs (n = 5 biological replicates). Baseline indicates normal high serum conditions. (B) Cell cycle analysis showing the effect of anaphase-promoting complex (APC), IL-6, TGF-β1, and/or EDC EVs on the proportion of atrial fibroblasts in the G0/G1 or S and G2M phases of the cell cycle (n = 5 biological replicates). Baseline indicates normal high serum conditions. (C) Effect of IL-6, TGF-β1, and/or EDC EVs on proteomic expression of cyclins that control progression of atrial fibroblasts through the cell cycle (n = 3 biological replicates). Baseline indicates normal high serum conditions. One-way ANOVA with individual-mean comparisons by Bonferroni’s multiple 2-tailed comparisons test.