Molecular events in MSC exosome mediated cytoprotection in cardiomyocytes

Figures

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  Figure 1. Characterization of exosomes. Western blots show enrichment of CD63 in exosomes while it is barely detected in whole cell lysates. Exosomes were plated onto a poly-L-ornithine coated glass slide and SEM images were acquired. The exosomes show a uniform size distribution of around 80 nm. Scale bar = 100 nm.

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  Figure 2. Monolayer cultures of cardiomyocytes were exposed to LPS (100 ng/ml) for 1 hour and then treated with MSC exosomes. LPS induced LOX-1 expression at 12 hrs of exposure. LPS also induced apoptosis (cleaved Caspase-3) and autophagy (LC3). Treatment with MSC exosomes decreased LOX-1, cleaved caspase-3 and LC-3 levels in cardiomyocytes stressed with LPS over a period of 6–12 hrs, but had no effect on expression of Beclin-1. LOX-1 levels were decreased with MSC exosomes. Data in mean ± SD, n = 4 *p < 0.05, vs LPS treatment based on 3 independent experiments. Full-length blots are presented in Supplementary Fig. 2.

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  Figure 3. Cell death (LDH release) and cell survival assays on cardiomyocytes. Cells were first treated with LPS (100 ng/ml) for 1 hr and then treated with MSC exosomes in presence of LPS. Treatment with MSC exosomes led to decrease in cell death and increase in cell survivability. Data as mean ± SD, n = 4. *p < 0.05 vs LPS treatment based on 3 independent experiments.

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  Figure 4. Cardiomyocyte spheroids were exposed to LPS (100 ng/ml) and then treated with MSC exosomes. Exposure to LPS reduced the spheroid diameter by >50% at 48 hrs. Treatment with exosomes rescued cardiomyocyte spheroid morphology in presence of LPS. *p < 0.05 vs LPS treatment based on 4 independent experiments.

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  Figure 5. Compared to the vesicle secreted protein database, MS analysis using FunRich32 analysis tool identified a number of similar and unique proteins secreted in MSC exosomes (A). Proteomic analysis identified different transcription factors in MSC exosomes (B). FunRich analysis revealed the different pathways which the secreted proteins modulate (C) and the processes which the exosomal proteins regulate (D). IPA analysis revealed the many this exosomal cargo modulates cellular proliferation, cell survival, and in recipient cells.

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  Figure 6. MS analysis of cardiomyocytes (CMs) using FunRich32 analysis tool: Differences in response to exosome treatment in cells can be attributed to the functional exosomal cargo in regulating gene expression such as changes in (A) expression of transcription factors (TFs) in LPS stressed cells or (B) LPS treated cells rescued by MSC exosomes. (C) Analysis of biological processes of proteins identified by MS in exosome rescued cardiomyocytes post LPS treatment. The processes presented are in comparison against LPS stimulated cardiomyocytes.

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  Figure 7. Network analysis: IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis)33 network analysis revealed upregulated signaling networks leading to cardiomyocytes death and cardiac fibrosis (A) and suppressed pathways leading to proliferation of cardiomyocytes, autophagy and fibroblast cell death (B). Exosome treatment of LPS stimulated cells upregulated signaling networks leading to increased cell viability and cell survival (C) while it decreased apoptotic signaling network (D).

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  Figure 8. MSC Exosomes provide significant cardioprotection against LPS stimulated stress responses. LPS treatment significantly increases pro-inflammatory LOX-1 expression along with cleaved caspase-3, and LC3. In spite of presence of LPS and continued stimulation of TLR4 LPS receptors, addition of MSC exosomes reverses the effects of LPS by downregulating expression of LOX-1, caspase-3 and LC3, thereby decreasing apoptosis and autophagy.