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Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice
Published on: April 10, 2019
Mesenchymal stem cell-derived exosomes ameliorate cardiomyocyte apoptosis in hypoxic conditions through microRNA144
Zhuzhi Wen1,2,3, Zun Mai3,4, Xiaolin Zhu1,2,3
1Department of Cardiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107 Yangjiang West Road, Guangzhou, 510120, China.
Background:
A growing body of evidence suggests that stem cell-derived exosomal microRNAs (miRNAs) could be a promising cardioprotective therapy in the context of hypoxic conditions. The present study aims to explore how miRNA-144 (miR-144), a miRNA contained in bone marrow mesenchymal stem cell (MSC)-derived exosomes, exerts a cardioprotective effect on cardiomyocyte apoptosis in the context of hypoxic conditions and identify the underlying mechanisms.
Methods:
MSCs were cultured using the whole bone marrow adherent method. MSC-derived exosomes were isolated using the total exosome isolation reagent and confirmed by nanoparticle trafficking analysis as well as western blotting using TSG101 and CD63 as markers. The hypoxic growth conditions for the H9C2 cells were established using the AnaeroPack method. Treatment conditions tested included H9C2 cells pre-incubated with exosomes, transfected with miR-144 mimics or inhibitor, or treated with the PTEN inhibitor SF1670, all under hypoxic growth conditions. Cell apoptosis was determined by flow cytometry using 7-ADD and Annexin V together. The expression levels of the miRNAs were detected by real-time PCR, and the expression levels of AKT/p-AKT, Bcl-2, caspase-3, HIF-1α, PTEN, and Rac-1 were measured by both real-time PCR and western blotting.
Results:
Exosomes were readily internalized by H9C2 cells after co-incubation for 12 h. Exosome-mediated protection of H9C2 cells from apoptosis was accompanied by increasing levels of p-AKT. MiR-144 was found to be highly enriched in MSC-derived exosomes. Transfection of cells with a miR-144 inhibitor weakened exosome-mediated protection from apoptosis. Furthermore, treatment of cells grown in hypoxic conditions with miR-144 mimics resulted in decreased PTEN expression, increased p-AKT expression, and prevented H9C2 cell apoptosis, whereas treatment with a miR-144 inhibitor resulted in increased PTEN expression, decreased p-AKT expression, and enhanced H9C2 cell apoptosis in hypoxic conditions. We also validated that PTEN was a target of miR-144 by using luciferase reporter assay. Additionally, cells treated with SF1670, a PTEN-specific inhibitor, resulted in increased p-AKT expression and decreased H9C2 cell apoptosis.
Conclusions:
These findings demonstrate that MSC-derived exosomes inhibit cell apoptotic injury in hypoxic conditions by delivering miR-144 to cells, where it targets the PTEN/AKT pathway. MSC-derived exosomes could be a promising therapeutic vehicle to facilitate delivery of miRNA therapies to ameliorate ischemic conditions.
Insights
Bone marrow mesenchymal stem cell-derived exosomes deliver microRNA-144 (miR-144) to protect heart cells from hypoxic injury. This therapy targets the PTEN/AKT pathway, offering a promising treatment for ischemic conditions.
Area of Science:
- Cardiology
- Stem Cell Biology
- Molecular Biology
Background:
- Stem cell-derived exosomes show potential for cardioprotection under hypoxia.
- MicroRNAs (miRNAs) within exosomes are key mediators of this effect.
- This study focuses on miRNA-144 (miR-144) from bone marrow mesenchymal stem cell (MSC)-derived exosomes.
Purpose of the Study:
- To investigate the cardioprotective role of miR-144 delivered by MSC-exosomes.
- To elucidate the underlying molecular mechanisms in cardiomyocyte apoptosis under hypoxia.
- To identify the specific molecular targets of miR-144 in this process.
Main Methods:
- Isolation and characterization of MSC-derived exosomes.
- Culture of H9C2 cardiomyocytes under hypoxic conditions.
- Treatment with exosomes, miR-144 mimics/inhibitors, and PTEN inhibitors.
- Assessment of cell apoptosis, miRNA expression, and protein levels (PTEN, AKT/p-AKT).
Main Results:
- MSC-exosomes were internalized by H9C2 cells and reduced apoptosis.
- miR-144, enriched in exosomes, mediated this protective effect by targeting PTEN.
- Upregulation of p-AKT and downregulation of PTEN were observed, inhibiting apoptosis.
- PTEN inhibition mimicked the protective effects of miR-144.
Conclusions:
- MSC-derived exosomes deliver miR-144 to inhibit cardiomyocyte apoptosis via the PTEN/AKT pathway under hypoxic conditions.
- This highlights the therapeutic potential of exosome-mediated miRNA delivery for ischemic heart disease.
- MSC-exosomes represent a promising vehicle for miRNA-based cardioprotective therapies.
