Butyrate-preconditioned human adipose-derived stem cell-conditioned medium enhances myocardial perfusion after infarction

Figures

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  Fig. 1. Transcriptomic analysis reveals functional changes in hASCs upon BA activation. a) Schematic representation of the experimental protocol for BA activation. hASCs were cultured with or without 1 mM BA in POWEREDBY10 medium supplemented with 3.0 pg/mL TNF-α and 10.0 pg/mL IFN-γ for 72 h. b) Heatmap showing differentially expressed genes (DEGs) among control (ASC), treated with BA (ASC-BA), treated with TNF-α and IFN-γ (ASC-TNIF), and treated with BA, TNF-α, and IFN-γ (ASC-BA-TNIF) (n = 3 per group) groups. Gene expression values are normalized and clustered. c) Volcano plot representing DEGs in between ASC-TNIF and ASC-BA-TNIF. Genes upregulated significantly in ASC-BA-TNIF are shown in yellow, and downregulated genes are in blue (adjusted p < 0.05, |log2 fold change| > 1). d) Gene Ontology (GO) enrichment analysis of upregulated genes in ASC-BA-TNIF.

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  Fig. 2. BA activation enhances the secretion of cytokines and chemokines in the hASC-conditioned medium (CM). a) Schematic representation of the CM collection protocol. hASCs were cultured with or without 1 mM BA in POWEREDBY10 medium supplemented with 3.0 pg/mL TNF-α and 10.0 pg/mL IFN-γ for 72 h. The medium was replaced with DMEM, and cells were incubated for an additional 48 h (wash-out phase). The resulting supernatants were collected and defined as ASC-CM (control) and ASC-BA-CM (BA-treated). b) Cytokine and chemokine levels in ASC-CM (blue circles) and ASC-BA-CM (red circles) as measured by cytokine/chemokine array (n = 3 per group). Asterisks indicate statistically significant differences (∗p < 0.05, unpaired t-test).

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  Fig. 3. Wound healing assay demonstrates enhanced endothelial repair by ASC-BA-CM. a) Representative images of a wound healing assay using HUVECs. A scratch was made in confluent HUVEC monolayers, followed by treatment with either ASC-CM or ASC-BA-CM. Images were captured at 0 and 24 h post-treatment. The dashed lines indicate the area of the initial scratch. In a separate co-culture system, HUVECs and THP-1 were cultured and subjected to the same assay. The bar graph quantifies the space occlusion ratio (percentage of wound closure relative to 0 h) in each group (n = 12 per group). Data are shown as mean ± SD. ∗∗p < 0.01 (unpaired t-test). b) Correlation analysis between chemokine concentrations in each CM sample and the space occlusion ratio. Pearson's correlation coefficient (r) and significance level (p) are indicated. ∗p < 0.05 was considered statistically significant.

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  Fig. 4. Intravenous administration of ASC-BA-CM promotes angiogenesis and macrophage recruitment in a sponge implantation model. a) Representative macroscopic images of sponge implants harvested on day 4 after intravenous administration of DMEM (medium), ASC-CM, or ASC-BA-CM (0.3 mL/day for sequential 3 days). The bar graph shows the hemoglobin concentration in the sponge tissues as an index of vascularization (n = 4–6 per group). ∗∗p < 0.01 (one-way ANOVA followed by Bonferroni post hoc test). b) Representative immunofluorescence images of sponge sections stained for CD105 (green, endothelial marker) and F4/80 (red, macrophage marker). Merged images show colocalization. Scale bar = 20 μm. c) Quantification of total cell number, macrophage ratio, and absolute macrophage number in sponge tissues analyzed by cytospin and immunocytochemistry. Data are presented as mean ± SD (n = 5 per group). ∗p < 0.05 (one-way ANOVA followed by Bonferroni post hoc test).

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  Fig. 5. Intravenous administration of ASC-BA-CM improves myocardial perfusion in a rat model of myocardial infarction. a) Representative SPECT/CT images of rats 28 days after intravenous administration of DMEM (control), ASC-CM, or ASC-BA-CM. Each image set includes four views: upper left (merged SPECT/CT from dorsal view), upper right (coronal SPECT), lower left (sagittal SPECT), and lower right (transversal SPECT). Myocardial perfusion levels are shown using a color scale. b) Serial SPECT images from the same animals acquired before treatment (Day 0, left side) and after 28 days of treatment (Day 28, right side). Each set displays coronal (top) and transversal (bottom) views to visualize perfusion recovery over time. c) Quantification of myocardial perfusion recovery based on the ratio of radioactive uptake (RI ratio) on Day 28 compared to Day 0. Data are presented as mean ± SD (n = 5 per group). ∗p < 0.05 (one-way ANOVA followed by Bonferroni post hoc test).

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  Fig. 6. Proposed schematic summary of observed effects and hypothesized mechanisms of ASC-BA-CM in myocardial perfusion improvement. BA preconditioning may enhance hASC activity, leading to increased paracrine secretion of cytokines and chemokines. The resulting conditioned medium (ASC-BA-CM) was demonstrated to improve myocardial perfusion following infarction. Based on these findings, ASC-BA-CM is hypothesized to contribute to endothelial repair and angiogenesis, as well as modulate immune responses, possibly through the polarization of macrophages. These potential mechanisms remain to be verified in future studies. Solid box indicates observed effects; dashed box and dashed arrows indicate hypotheses.