Extracellular Vesicles Derived From Human Umbilical Cord Mesenchymal Stem Cells Protect Against DOX-Induced Heart Failure Through the miR-100-5p/NOX4 Pathway

Abstract

The end result of a variety of cardiovascular diseases is heart failure. Heart failure patients' morbidity and mortality rates are increasing year after year. Extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (HucMSC-EVs) have recently been discovered to be an alternative treatment for heart failure, according to recent research. In this study, we aimed to explore the underlying mechanisms in which HucMSC-EVs inhibited doxorubicin (DOX)-induced heart failure in AC16 cells. An miR-100-5p inhibitor and an miR-100-5p mimic were used to transfect HucMSCs using Lipofectamine 2000. HucMSC-EVs were isolated and purified using the ultracentrifugation method. AC16 cells were treated with DOX combined with HucMSC-EVs or an EV miR-100-5-p inhibitor or EV miR-100-5-p mimic. ROS levels were measured by a flow cytometer. The levels of LDH, SOD, and MDA were measured by biochemical methods. Apoptotic cells were assessed by a flow cytometer. Cleaved-caspase-3 and NOX4 protein expression were determined by Western blot. The experiment results showed that HucMSC-EVs inhibited DOX-induced increased levels of ROS, LDH, and MDA, and decreased levels of SOD which were reversed by an EV miR-100-5-p inhibitor, while EV miR-100-5-p mimic had a similar effect to HucMSC-EVs. At the same time, HucMSC-EV-inhibited DOX induced the increases of apoptotic cells as well as NOX4 and cleaved-caspase-3 protein expression, which were reversed by an EV miR-100-5-p inhibitor. Furthermore, the NOX4 expression was negatively regulated by miR-100-5p. Overexpression of NOX4 abolished the effects in which HucMSC-EVs inhibited DOX-induced ROS, oxidative stress, and apoptosis increases. In conclusion, these results indicate that HucMSC-EVs inhibit DOX-induced heart failure through the miR-100-5p/NOX4 pathway.

Keywords: NOX4; extracellular vesicles; heart failure; human umbilical cord mesenchymal stem cells; miR-100-5p; oxidative stress.

FIGURE 1

Isolation and identification of HucMSC-EVs. (A) The surface markers CD44 and CD105 of HucMSCs were identified by flow cytometry. (B) The morphology of purified HucMSC-EVs was observed by transmission electron microscopy: scale bar: 200 nm. (C) The markers CD81 and TSG101 of HucMSC-EVs were detected by Western blot. (D) HucMSC-EV endocytosis traced by PKH67 was observed by a laser scanning microscope in AC16 cells.

FIGURE 2

HucMSC-EVs inhibit DOX-induced oxidative stress and apoptosis. AC16 cells were treated with 2 µmol/L DOX and HucMSC-EVs at different concentrations (0, 50, 100, and 200 μg/ml) for 24 h. (A) ROS levels were measured by flow cytometry. (B) The percentages of apoptotic cells were assessed by flow cytometry. Quadrant 1 represented dead cells. Quadrant 2 represented late apoptotic cells. Quadrant 3 represented early apoptotic cells. Quadrant 4 represented normal cells. (C–E) The levels of LDH (C), SOD (D), and MDA (E) were measured with biochemistry methods. (F) Western blot analysis of cleaved-caspase-3. ^*** p < 0.001 vs. vehicle, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. 2 μmol/L_DOX + 0 μg/ml_HucMSC-EVs, ⁺ p < 0.05, ⁺⁺ p < 0.01 vs. 2 μmol/L_DOX + 50 μg/ml_HucMSC-EVs.

FIGURE 3

HucMSC-EVs inhibits NOX4 expression. AC16 cells were treated with 2 µmol/L DOX and HucMSC-EVs at different concentrations (0, 50, 100, and 200 μg/ml) for 24 h. (A) qRT-PCR and (B) Western blot were performed to detect NOX2 and NOX4 expression, respectively. ^*** p < 0.001 vs. vehicle, ^# p < 0.05, ^## p < 0.01 vs. 2 μmol/L_DOX + 0 μg/ml_HucMSC-EVs, ⁺ p < 0.05 vs. 2 μmol/L_DOX + 50 μg/ml_HucMSC-EVs. (C–D) AC16 cells were treated with 2 µmol/L DOX and HucMSC-EVs at a concentration of 100 μg/ml for different times (0, 6, 12, 24, and 48 h). (C) qRT-PCR and (D) Western blot were used for the detection of NOX4 expression. ^*** p < 0.001 vs. Vehicle, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. 0 h, ⁺ p < 0.05 vs. 12 h.

FIGURE 4

Inhibition of EV miR-100-5-p reverses the effects in which HucMSC-EVs inhibit DOX-induced oxidative stress and apoptosis. (A) HucMSCs were transfected with miR-100-5p inhibitor (Inhibitor) or miR-100-5p mimic (Mimic). MiR-100-5p expression was determined by Q-PCR. (B) HucMSCs were transfected with miR-100-5p inhibitor or miR-100-5p mimic. After HucMSC-EVs were extracted, EV miR-100-5-p was determined by Q-PCR. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. NC. (C–H) AC16 cells were treated with 2 µmol/L DOX and EV miR-100-5p inhibitor (Inhibitor-EVs) or EV miR-100-5-p mimic (Mimic-EVs) at a concentration of 100 µg/ml. (C) ROS levels were measured by flow cytometry. (D) Apoptosis was determined by flow cytometry. Quadrant 1 represented dead cells. Quadrant 2 represented late apoptotic cells. Quadrant 3 represented early apoptotic cells, and Quadrant 4 represented normal cells. (E–G) LDH, SOD, and MDA levels were measured with biochemical assay. (H) NOX4 and cleaved-caspase-3 expression were examined by Western blot. ^*** p < 0.001 vs. control, ^## p < 0.01 vs. 2 µmol/DOX, ⁺ p < 0.05, ⁺⁺ p < 0.01 vs. 2 µmol/L DOX + NC-EVs.

FIGURE 5

Overexpression of NOX4 abolishes those effects in which HucMSC-EVs inhibit DOX-induced oxidative stress and apoptosis. 293T cells were co-transfected with either pGL3-NOX4-WT, miR-100-5p inhibitor (Inhibitor) and miR-100-5p Mimic, and pGL3-NOX4-MUT, mimic (Mimic) or pGL3-NOX4-MUT, miR-100-5p inhibitor (Inhibitor) and miR-100-5p mimic (Mimic). (A) Luciferase activity was quantified using a luminometer. ^** p < 0.01, ^*** p < 0.001 vs. WT + NC. (B) NOX4 mRNA was examined by qRT-PCR. (C) NOX4 protein expression was detected by Western blot. ^** p < 0.01 vs. NC. (D) AC16 cells were transfected with oeNOX4. NOX4 mRNA was examined by qRT-PCR. (E) AC16 cells were transfected with oeNOX4. NOX4 protein was detected by Western blot. ^*** p < 0.001 vs. vector. (F–K) AC16 cells were transfected with oeNOX4 for 24 h and then treated with 2 µmol/L DOX and 100 µg/ml HucMSC-EVs for 24 h. (F) ROS levels were measured by flow cytometry. (G) Apoptosis was measured by flow cytometry. Quadrant 1 represented dead cells. Quadrant 2 represented late apoptotic cells. Quadrant 3 represented early apoptotic cells, and Quadrant 4 represented normal cells. LDH (H), SOD (I), and MDA (J) were measured by biochemical assay. (K) NOX4 and cleaved-caspase-3 expression were determined by Western blot. ^*** p < 0.001 vs. vehicle, ^## p < 0.01 vs. 2 μmol/L_DOX + vehicle, ⁺⁺⁺ p < 0.001 vs. 2 µmol/L_DOX + HucMSC-EVs + vector.