Inhibiting Extracellular Vesicle Release from Human Cardiosphere Derived Cells with Lentiviral Knockdown of nSMase2 Differentially Effects Proliferation and Apoptosis in Cardiomyocytes, Fibroblasts and Endothelial Cells In Vitro

Figures

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  Fig 1. Characterization of human cardiosphere-derived cells (CDCs). (A-C) Representative fluorescence activated cell sorting (FACS) plots of CDCs stained with CD90, CD105 and CD45 (plotted in red) and their respective isotype controls (plotted in black). hCDCs express high levels of mesenchymal markers CD90 and CD105 and are negative for leukocyte common antigen marker, CD45. (D) hCDCs stain positive for cardiac transcription factor GATA4 and Nkx2.5 and negative for Troponin T. Scale bar 200μm.

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  Fig 2. Characterization of CDC-derived exosomes. (A) Nanoparticle analysis of particle distribution profile shows human CDC derived exosomes largely range between 65 and 173nm in diameter. (B) Immunoblot analysis of exosomal markers CD63 and HSP90 in CDC derived exosomes and (C) non-exosomal makers, cytochrome c and EEA1, in exosomes and corresponding CDC whole-cell lysate (15μg/well). (D) Transmission electron microscope image of human CDC-secreted exosome at 100 keV. Negative contrast staining showing typical exosome morphology.

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  Fig 3. Differential Effect of CDC-derived exosomes are internalized by cardiomyocytes, cardiac fibroblasts and HUVECs. Isolated CDC-derived exosomes labeled with Exo-Red (nucleic acid label) were incubated with (A) cardiomyocytes, (B) cardiac fibroblasts, and (C) HUVECs for 6 hours and imaged to show cellular uptake. The frequency of hCDC-derived exosome uptake was lower for cardiomyocytes than endothelial cells or fibroblasts. Scale bar 50μm.

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  Fig 4. Stable transduced hCDC-nSMase2 lentiviral knockdown lines block exosome secretion. (A) Bradford assay and (B) immunoblot analysis of CD63 of exosomes from CDCs transduced with lentiviral vectors for scrambled or nSMase2-specific shRNAi show nSMase2 knockdown results in significantly less release of exosomes from human CDCs (n = 3). *p<0.01 using two-tailed Student’s t test. Data are presented as mean ± SEM.

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  Fig 5. nSMase2 lentiviral integration does not change CDC survival, proliferation or gene expression. Nontransduced CDCs and nSMase2 shRNAi lentiviral infected CDCs show no significant difference in (A) survival assessed with calcien staining, (B) proliferation assessed with BrDU+ pulse or (C) surface marker expression (CD90, CD105 and CD45 plotted in red and their respective isotype controls plotted in black).

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  Fig 6. hCDC-derived exosomes promote HUVEC migration without affecting proliferation. (A) The migratory response of HUVECs following hCDC coculture was studied using a 4 hour transwell migration assay. HUVECs cocultured with lentiviral Scrambled control (Scr) CDCs had increased migration as hen compared to endothelial cells cocultured with lentiviral:nSMase2 KD CDCs (57 vs 41%) and media control (n = 3). (B) HUVECs cocultured with Scr CDCs and pulsed with BrdU demonstrated no change in proliferation compared to those cocultured with nSMase2 KD CDCs (% BrdU+ cells/total number of DAPI+ cells; n = 3). *p<0.05 by one-way ANOVA (Tukey’s post hoc test). Data are presented as mean ± SEM.

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  Fig 7. hCDC exosomes stimulate angiogenesis in a HUVEC angiogenesis assay. HUVECs cocultured with Scr CDCs for 24 hours show an increased number of branch points and a trend towards increased tube length as compared with HUVECs cocultured with nSMase2 KD CDCs in a 4 hour matrigel in vitro tube assay (n = 3). Scale bar 200μm. * p<0.001 using one-way AVOVA (Tukey’s post hoc test). Data are presented as mean ± SEM.

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  Fig 8. hCDC exosomes reduce proliferation of cardiac fibroblasts without affecting collagen gene expression. (A, B) Human quiesced cardiac fibroblasts cocultured with either Scr CDCs or KD CDCs showed no statistically significant difference in COL1 or COL3 gene expression by qRT-PCR following TGFβ stimulation (n = 3). Data normalized to unstimulated Scr CDCs. (C, D) Cardiac fibroblasts cocultured with Scr CDCs for 24 hours showed a significant reduction in cellular proliferation as assessed by BrdU staining (5 hour pulse) when compared with (C, E) cardiac fibroblasts cocultured with nSMase2 KD CDCs and media control. n = 3. Scale bar 200μm. *p<0.05 using one-way AVOVA (Tukey’s post hoc test). Data are presented as mean ± SEM. White arrows highlight cells that co-stain for DAPI and BrdU.

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  Fig 9. hCDC-derived exosomes significantly decrease cardiomyocyte programed cell death and cause a trend toward increased proliferation of cardiomyocytes in vitro at one week in culture. (A, C) Primary mouse neonatal cardiomyocytes (mCM) (P1-3) cocultured with Scr CDCs exhibited a trend toward a higher percentage of BrdU+ cells at one week following a 5 day pulse when compared with (B, C) mCM cocultured with nSMase2 KD CDCs (n = 4; p = 0.19 using one-way ANOVA with Tukey’s post hoc test). Scale bar 50μm. (D, F) mCM cocultured with Scr CDCs exhibited fewer terminal deoxynucleotidyl transferase nick end labeling (TUNEL)-positive nuclei at one week when compared with (E, F) mCM cocultured with nSMase2 KD CDCs and media control (n = 4). The magnitude of the effect of CDC exosomes on myocyte apoptosis was nearly two orders of magnitude larger than proliferation assessed by BrdU over this time frame. Scale bar 10μm. *p<0.001 using one-way AVOVA (Tukey’s post hoc test). Data are presented as mean ± SEM.