Extracellular Vesicles Released after Doxorubicin Treatment in Rats Protect Cardiomyocytes from Oxidative Damage and Induce Pro-Inflammatory Gene Expression in Macrophages

Figures

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  Figure 1. Timeline of DOXO treatment and blood collection in Wistar rats (A). Representative picture of Western blot of Bax, cleaved caspase-3, pro caspase-3, and GAPDH from heart tissues of saline-treated (SAL) and DOXO-treated (DOXO) rats (B). Fold change of band intensities of Bax relative to GAPDH (C), cleaved caspase-3 relative to GAPDH (D), pro caspase-3 relative to GAPDH (E) in SAL (n = 5) and DOXO (n = 5) groups. Concentration of NT-proBNP in serum of SAL (n = 5) and DOXO (n = 5) groups (F); * p < 0.05 vs. SAL. The effect of Doxorubicin on left ventricular function and cardiac sympathovagal balance (G–L). (G). LVEF (%), (H). LVFS (%), (I). E/A ratio, (J). LF (normalized unit), (F). HF (normalized unit), (G). LF/HF ratio; DOXO, doxorubicin; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening, E/A, early filling to atrial filling velocities ratio; LF/HF, low-frequency component to high-frequency component ratio. Data are presented as the mean ± SEM; * p < 0.05 vs. baseline; † p < 0.05 vs. SAL at that period of time.

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  Figure 2. Diagram illustrating isolation of serum Evs using size exclusion chromatography (SEC) including void volume and collection of flow though fractions for Western blot analysis (A), Western blot of potential EV markers (CD63, CD9, and CD81), as well as a non-EV protein—ERp72—in fractions 5 to 11 from SEC of SAL and DOXO-rat sera (B).

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  Figure 3. Representative graphs of size distributions of SAL_EVs (A) and DOXO_EVs (B) size distribution. Blue line represents the averaged data, whereas red line represents the SE. The mean size ± SE of SAL_EVs and DOXO_EVs (C)The average EV concentration ± SE of SAL_EVs (n = 5) and DOXO_EVs (n = 5) (D). * p < 0.05.

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  Figure 4. Levels of ROS production measured by DCFDA assay (A) and PO-1 assay (B) in H9c2 cells pre-incubated with SAL_EVs or DOXO_EVs for 24 h followed by PBS or 100 μM H2O2 treatment for 10 min; * p < 0.05 vs. no EV, no H2O2 treated control (PBS), ‡ p < 0.05 vs. SAL_EVs 5 Levels of Gpx1 (C), Cat (D) and Sod2 (E) gene expression in H9c2 cells incubated with SAL_EVs or DOXO_EVs for 24 h; SAL_EVs 5 = H9c2 cells pre-incubated with SAL_EVs 5 × 109 particles/mL; SAL_EVs 10 = H9c2 cells pre-incubated with SAL_EVs 10 × 109 particles/mL; DOXO_EVs 5 = H9c2 cells pre-incubated with DOXO_EVs 5 × 109 particles/mL; DOXO_EVs 10 = H9c2 cells pre-incubated with DOXO_EVs 10 × 109 particles/mL; * p < 0.05 vs EVs from the same group at concentration 5 × 109 particles/mL, † p < 0.05 vs SAL_EVs 10 × 109 particles/mL.

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  Figure 5. Percentage of cell death measured by LDH cytotoxicity assay in H9c2 cells pre-incubated with PBS, SAL_EVs or DOXO_EVs followed by 100 or 200 μM H2O2 exposure for 24 h. SAL_EVs 10 = H9c2 cells pre-incubated with SAL_EVs 10 × 109 particles/mL, SAL_EVs 20 = H9c2 cells pre-incubated with SAL_EVs 20 × 109 particles/mL; DOXO_EVs 10 = H9c2 cells pre-incubated with DOXO_EVs 10 × 109 particles/mL; DOXO_EVs 20 = H9c2 cells pre-incubated with DOXO_EVs 20 × 109 particles/mL; * p < 0.05 vs. no EV treated control (PBS), † p < 0.05 vs. SAL_EVs 10 × 109 particles/mL; ‡ p < 0.05 vs. DOXO_EVs 10 × 109 particles/mL.

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  Figure 6. Levels of ROS production measured by DCFDA assay (A) and PO-1 assay (B) in NR8383 cells pre-incubated with SAL_EVs or DOXO_EVs for 24 h; SAL_EVs 5 = NR8383 cells pre-incubated with SAL_EVs 5 × 109 particles/mL; SAL_EVs 10 = NR8383 cells pre-incubated with SAL_EVs 10 × 109 particles/mL; DOXO_EVs 5 = NR8383 cells pre-incubated with DOXO_EVs 5 × 109 particles/mL; DOXO_EVs 10 = NR8383 cells pre-incubated with DOXO_EVs 10 × 109 particles/mL; * p < 0.05 vs. no EV (PBS), † p < 0.05 vs. DOXO_EVs 5 × 109 particles/mL. Levels of Il6 (C), Tnf (D), Arg1 (E), Tgfb (F) gene expression in NR8383 cells incubated with SAL_EVs or DOXO_EVs for 24 h; * p < 0.05 vs. PBS, † p < 0.05 vs. SAL_EVs 5 × 109 particles/mL, ‡ p < 0.05 vs. DOXO_EVs 10 × 109 particles/mL.