Extracellular vesicles derived from microRNA-150-5p-overexpressing mesenchymal stem cells protect rat hearts against ischemia/reperfusion

Figures

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  Figure 1. TXNIP is highly expressed in the rat model of I/R. (A) The preoperative, intraoperative and postoperative ST segments (mV) in sham-operated rats (n = 12) (left) and in rats with I/R (n = 72) (right); * p < 0.05 vs. the preoperative ST segment; # p < 0.05 vs. the intraoperative ST segment. (B) TXNIP expression in myocardium determined by RT-qPCR; * p < 0.05 vs. the sham group (sham-operated rats). (C) TXNIP protein expression in myocardium normalized to GAPDH determined by Western blot analysis; * p < 0.05 vs. the sham group (sham-operated rats). (D) The positive expression of TXNIP in myocardium identified by immunohistochemistry (400 ×); * p < 0.05 vs. the sham group (sham-operated rats). Measurement data were presented as mean ± standard deviation. Comparison between two groups was analyzed by unpaired t-test. Comparison among multiple groups was analyzed by one-way analysis of variance, followed by Tukey’s post hoc test. n = 12.

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  Figure 2. TXNIP knockdown impedes myocardial remodeling of rats with I/R. (A) TXNIP expression in myocardium determined by RT-qPCR. (B) TXNIP protein expression in myocardium normalized to GAPDH determined by Western blot analysis. (C) The myocardial infarct size detected by TTC staining. (D) The myocardial collagen detected by Masson's staining (200 ×). (E) The cardiomyocyte apoptosis detected by TUNEL staining (400 ×). (F) The protein expression of apoptosis-related factors (c-Jun, Bax and Bcl-2) normalized to GAPDH determined by Western blot analysis. * p < 0.05 vs. the sham group (sham-operated rats); # p < 0.05 vs. the I/R + sh-NC group (I/R rats treated with sh-NC). Measurement data were presented as mean ± standard deviation. Comparison between two groups was analyzed by non-paired t-test. n = 12.

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  Figure 3. TXNIP is the target of miR-150-5p. (A) The binding sites between miR-150-5p and TXNIP as predicted by microrna.org. (B) The relative luciferase activity determined by dual-luciferase reporter gene assay. (C) The miR-150-5p expression in myocardium determined by RT-qPCR, normalized to U6; * p < 0.05 vs. the sham group (sham-operated rats); n =12. (D) The mRNA expression of TXNIP in myocardium determined by RT-qPCR, normalized to GAPDH. (E) The protein expression of TXNIP in myocardium normalized to GAPDH determined by Western blot analysis. (F) The expression of miR-150-5p in cardiomyocytes in response to miR-150-5p-agomir and miR-150-5p-antagomir determined by RT-qPCR. * p < 0.05 vs. the agomir-NC group (I/R rats treated with agomir-NC); # p < 0.05 vs. the antagomir-NC group (I/R rats treated with antagomir-NC). Measurement data were presented as mean ± standard deviation. Comparison between two groups was analyzed by unpaired t-test. The cell experiment was repeated 3 times independently.

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  Figure 4. Cardiomyocyte apoptosis is suppressed by MSCs-derived EVs carrying miR-150-5p. (A) The images captured under transmission electron microscopy. (B) EV size measured by nanoparticle tracking analysis. (C) The expression of EV markers (CD9, CD63, Alix and GRP94) detected by Western blot analysis. (D) EV internalization detected by immunohistochemistry (400 ×). (E) PKH67-staining (FITC wavelength) in neonatal cardiomyocytes detected by flow cytometric analysis. (F) The expression of miR-150-5p in MSCs determined by RT-qPCR. (G) The expression of miR-150-5p in EVs determined by RT-qPCR. (H) neonatal cardiomyocyte apoptosis detected by TUNEL staining (200 ×). (I) The protein expression of apoptosis-related factors (c-Jun, Bax and Bcl-2) normalized to GAPDH determined by Western blot analysis. (J) The expression of miR-150-5p and TXNIP in neonatal cardiomyocytes determined by RT-qPCR. (K) TXNIP expression in neonatal cardiomyocytes normalized to GAPDH determined by Western blot analysis. (L) Transwell co-culture system under hypoxia/hypoglycemia condition. (M) Neonatal cardiomyocyte apoptosis detected by TUNEL staining (200 ×); * p < 0.05 vs. the agomir-NC group (cells treated with…

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  Figure 5. The EVs-loaded with miR-150-5p/TXNIP mediates myocardial remodeling after I/R. (A) The transduction efficiency detected by RT-qPCR. (B) The myocardial infarct size detected by TTC staining. (C) The myocardial collagen detected by Masson's staining (200 ×). (D) The cardiomyocyte apoptosis detected by TUNEL staining (400 ×). (E) The protein expression of apoptosis-related factors (c-Jun, Bax and Bcl-2) normalized to GAPDH determined by Western blot analysis. * p < 0.05 vs. the I/R + EVagomir-NC group (I/R rats treated with EVagomir-NC); # p < 0.05 vs. the I/R + EVmiR-150-5p-agomir group (I/R rats treated with EV miR-150-5p-agomir). Measurement data were presented as mean ± standard deviation. Comparison among multiple groups was analyzed by one-way analysis of variance, followed by Tukey’s post hoc test. n = 12.

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  Figure 6. The schematic diagram depicts molecular basis underlying EVs derived from MSCs on I/R by transferring miR-150-5p. MSCs can secrete EVs to transfer miR-150-5p into cardiomyocytes, which further internalize EVs to release miR-150-5p and miR-150-5p protects against I/R by downregulating TXNIP and inhibiting myocardial remodeling.