Diabetic state of human coronary artery endothelial cells results in altered effects of bone mesenchymal stem cell‐derived extracellular vesicles

Figures

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  FIGURE 1. HBMSC‐EV characterizations. (a) NanoSight particle analysis showing size distribution of EVs with corresponding concentrations. (b) Electron microscopy image of EVs (scale bar = 200 nm; magnification 54,800×). (c) Western blot of the HBMSC‐EV demonstrated the presence of transmembrane proteins CD81 and CD9, cytosolic protein Alix and GAPDH. HSP70, a promiscuous cytosolic protein, was not found. Albumin was not identified, which demonstrated purity of the HBMSC‐EV isolation. For western blot characterization of extracellular vesicles, approximately 10 μg of protein was loaded per lane and was interpreted only qualitatively, thus no quantification was done.

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  FIGURE 2. Outputs from ShinyGO showing basal differences in metabolism, inflammation, RNA processing and biogenesis pathways in HCAEC (n = 4) and DM‐HCAEC (n = 4). (a) Hierarchal clustering tree of pathways up‐regulated in DM‐HCAEC compared to HCAEC. Phosphorus metabolic process and myeloid leukocyte mediated immunity were notably upregulated, with p‐values of 1.1 × 10−14 and 1.1 × 10−14, respectively. The statistical significance of these pathways are noted in the p‐values to the left of the respective pathway. Proteins used for the pathway analysis had un‐adjusted p‐values < 0.055. (b) Hierarchal clustering tree of pathways down‐regulated in DM‐HCAEC compared to HCAEC. RNA processing and cellular biogenesis pathways were down‐regulated in DM‐HCAEC.

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  FIGURE 3. Outputs from ShinyGO showed that HBMSC‐EV treatment of HCAEC (n = 4) resulted in increased expression of RNA processing and decreased phosphorus metabolic processes and inflammatory pathways compared to that of vehicle treatment only (n = 4). (a) Hierarchal clustering tree of pathways up‐regulated in HCAEC after HMBSC‐EV treatment. The statistical significance of these pathways are denoted on the left of the pathway text. Significant proteins used for pathway analyses had un‐adjusted p‐values < 0.055. (b) Hierarchal clustering tree with associated p‐values of pathways down‐regulated in HCAEC after HBMSC‐EV treatment.

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  FIGURE 4. Outputs from ShinyGO showed that HBMSC‐EV treatment of DM‐HCAEC (n = 4) resulted in mixed up and down‐regulation of RNA processing pathways and no changes in phosphorus metabolic processes or inflammatory pathways compared to that of vehicle treatment only (n = 4). This may be due to baseline differences between HCAEC and DM‐HCAEC. (a) Hierarchal clustering tree of pathways up‐regulated in DM‐HCAEC after HBMSC‐EV treatment. The p‐values denoting the statistical significance of these pathways is to the left of the pathway. Regulation of apoptosis and positive regulation of various metabolic processes were up‐regulated, as well as RNA splicing (though to a lesser extent to that of HCAEC). (b) Hierarchal clustering tree with pathways down‐regulated in DM‐HCAEC after HBMSC‐EV treatment. The RNA processing pathway was found to be decreased here, in addition to being up‐regulated, representing a mixed response. No phosphorus metabolic processes or inflammatory pathways were identified here.

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  FIGURE 5. Proteins related to key metabolic pathways and antioxidant regulation were found to be altered in DM‐HCAEC (n = 5) compared to HCAEC (n = 5) on western blot (right panel), where 10 μg of protein was loaded per well. This data represents findings from one donor per cell type. The Shapiro–Wilk and Mann–Whitney statistical tests were used for analysis. (a) Increase in protein expression of key pathways demonstrate activation of both starvation/stress (AMPK) and proliferative (ERK, mTOR) signalling in DM‐HCAEC. (b) There was an overall decreased expression of multiple antioxidant proteins (GPX1, SOD2, NRF2) in DM‐HCAEC, which may explain the state of increased oxidative stress in DM‐HCAEC.