Hypoxic preconditioned mesenchymal stem cell-derived exosomes alleviate oxidative stress-induced cardiomyocyte apoptosis through miR-486-5p

Abstract

BACKGROUND

Emerging evidence indicates that hypoxic preconditioning boosts the antioxidant and anti-apoptotic capacities of mesenchymal stem cell-derived exosomes; however, the specific mechanisms remain incompletely elucidated. This study explored the impact of hypoxia-preconditioned mesenchymal stem cell-derived exosomes (hypo-Exos) vs normoxic counterparts on the apoptotic response in cardiomyocytes triggered by oxidative stress.

AIM

To determine whether and how hypoxic preconditioning augments the cardioprotective efficacy of hypo-Exos against oxidative stress-induced cardiomyocyte apoptosis.

METHODS

H9C2 cardiomyocytes were treated with hydrogen peroxide (H₂O₂) to induce oxidative injury. Assessments of cell viability, oxidative biomarkers, and apoptotic activity were conducted to evaluate the therapeutic efficacy of hypo-Exos and normoxic counterparts. High-throughput sequencing was performed to identify potential target microRNAs (miRNAs). Luciferase reporter assays were conducted to confirm selected miRNAs binding to target genes. Hypo-Exos loaded with selected miRNAs antagomirs or negative controls were administered to H₂O₂-treated H9C2 cells to validate the downstream signaling pathways involved.

RESULTS

Hypo-Exos significantly enhanced cell viability, reduced oxidative stress, and inhibited apoptosis of cardiomyocytes. Hypoxic preconditioning significantly increased the expression of exosomal miR-486-5p, which directly targeted the phosphatase and tensin homolog. Additionally, hypo-Exos markedly activated the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Moreover, deletion of miR-486-5p in hypo-Exos counteracted the anti-apoptotic effects and suppressed PI3K/Akt pathway activation.

CONCLUSION

Hypoxic preconditioning augments anti-apoptotic properties of exosomes, primarily via miR-486-5p upregulation, which mediates its function by modulating the phosphatase and tensin homolog/PI3K/Akt axis.

Key Words: Mesenchymal stem cells; Hypoxic preconditioning; Exosomes; miR-486-5p; Oxidative stress; Apoptosis

Core Tip: Hypoxic preconditioning enhances the therapeutic properties of mesenchymal stem cell-derived exosomes with elevated miR-486-5p, which directly inhibits phosphatase and tensin homolog to activate the phosphoinositide 3-kinase/protein kinase B signaling, thereby halting oxidative stress-driven cardiomyocyte apoptosis. This work reveals a microRNA-mediated mechanism that allows exosomes to have powerful cardioprotective effects, suggesting a cell-free therapeutic strategy for treating oxidative-related cardiac injury.

INTRODUCTION

Oxidative stress arises when intracellular or tissue levels of reactive oxygen species (ROS) exceeds the neutralizing capacity of the antioxidant system, leading to redox imbalance[1]. The increase in ROS is the main cause of cardiovascular diseases, leading to lipid peroxidation, protein damage, and DNA mutations[2]. Studies demonstrate that oxidative stress is significantly elevated in patients with heart failure, damaging cardiomyocytes and causing mitochondrial dysfunction, apoptosis, and necrosis, thereby impairing cardiac function[3]. Furthermore, oxidative stress activates multiple signaling pathways such as mitogen-activated protein kinases and Ras to promote fibrosis and inflammation, and remodels the extracellular matrix, thereby accelerating ventricular remodeling and reducing cardiac performance[4]. Therefore, it is crucial to inhibit oxidative stress and apoptosis in cardiovascular diseases.

Mesenchymal stem cells (MSCs) have garnered significant interest as promising therapeutic agents because of their multilineage differentiation, tissue repair, and immune modulation[5]. The application of MSCs in cardiovascular disease research and clinical therapy has rapidly expanded[6]. Notably, human umbilical cord-derived MSCs (HUC-MSCs) are considered a superior treatment choice due to their accessibility, minimal immunogenic response, and robust self-renewal properties[7]. However, MSCs face challenges in therapeutic applications, including low survival rates, limited migration capabilities, and risk of immune rejection[8]. By contrast, MSC-derived exosomes offer improved capacities to traverse tissue barriers, selectively target injured cells, and reduce immune and ethical concerns, which are attributed to their small size and capacity to carry functional microRNAs (miRNAs), proteins, and lipids[9].

Hypoxic preconditioning is emerging as a widely acknowledged method for effective cell activation[10]. This technique simulates a low-oxygen environment and regulates gene expression linked to cell survival, metabolism, and antioxidant responses, thereby enhancing cellular resilience and repair mechanisms[11]. Studies suggest that hypoxic preconditioning enhances the antioxidant capacity of MSCs, reduces apoptosis, and promotes their migration to injured tissues[12]. Furthermore, it has been found that hypoxic preconditioning alters the composition of exosomes, increasing the loading of antioxidant miRNAs and anti-apoptotic factors, thereby further enhancing the practical efficacy[13]. However, the capacity of hypoxic preconditioned exosomes in inhibiting cardiomyocyte oxidative stress and apoptosis has not been studied.

miRNAs are short non-coding RNAs that modulate the expression of multiple genes and contribute to diverse biological processes[14]. In cardiovascular diseases, specific miRNAs can modulate oxidative stress responses and influence cardiomyocyte apoptosis, thereby affecting cardiac function[15]. Research has shown that exosomal miR-146a reduces cardiomyocyte apoptosis and protects against myocardial injury[16]. MSC-derived exosomes are abundant in a variety of miRNAs that can target essential cardiomyocyte genes, reducing oxidative stress and apoptosis and positively contributing to cardiovascular disease management[17]. miR-486-5p is a highly conserved, muscle-enriched miRNA implicated in multiple biological processes that protect against ischemia-reperfusion injury; regulates apoptosis; and participates in the regulation of cell proliferation, differentiation, and metabolic pathways in various tissues[18-21].

This study focused on exploring the effects of hypoxia-preconditioned MSC-derived exosomes (hypo-Exos) on inhibiting cell apoptosis and uncovering their underlying therapeutic mechanisms under conditions of heightened oxidative stress.

MATERIALS AND METHODS

Hypoxia exposure and identification of HUC-MSCs

HUC-MSCs were provided by the Precision Medical Center of Nanfang Hospital (Guangdong, China), with the informed consent of all participants and approved by the medical ethics committee of the hospital (Approval No. NFEC-202110-K17-01). HUC-MSCs were maintained in alpha-MEM (Shanghai Basal Media Technologies Co., Ltd., Shanghai, China) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (VivaCell, Shanghai, China) inside an incubator preserved at 37 °C with 5% CO₂. Once confluency reached approximately 80%, the cells were passaged into new flasks to promote continued proliferation. Only HUC-MSCs from passages 3 to 5 were selected for subsequent experiments. At nearly 80% confluency, the existing culture solution was replaced by BC-T4 complete medium containing 5% serum-free supplement (UltraGRO^TM-Advanced; Helios Biochemical Science and Technology Co., Ltd., Jiangsu, China) for 24 hours. Following this incubation, the HUC-MSCs were maintained either under normoxia (21% O₂) or exposed to hypoxic conditions (1% O₂) for 48 hours. The hypoxic environment was established using a hypoxic incubator (Thermo Fisher Scientific, Waltham, MA, United States). Phenotyping of the cells was conducted via flow cytometry (FACSCanto II; Becton Dickinson, San Diego, CA, United States), along with assessments of cellular morphology to identify HUC-MSCs. The analysis centered on multiple molecular markers such as cluster of differentiation 105 (CD105) (98013-2-RR; Proteintech, Wuhan, China), CD90 (98126-1-RR; Proteintech), CD73 (65162-1-Ig; Proteintech), CD45 (98117-1-RR; Proteintech), and CD34 (98145-4-RR; Proteintech).

Isolation of exosomes

Following a 48-hour incubation period, the conditioned media from HUC-MSCs were harvested and purified through deep filtration (Pall Corporation, Port Washington, NY, United States) operated at a 10 mL/minute permeation velocity. Subsequently, an ultrafiltration process was conducted using a tangential flow filtration membrane package with a 100-kDa cutoff (Pall Corporation). During this filtration, the transmembrane pressure was kept below 10 psi, with a membrane throughput of 10 mL/minute and an inflow rate of 120 mL/minute consistently upheld. Once concentration reached ninefold, a buffer exchange was performed using phosphate-buffered saline (PBS) at six times the volume. The exosome solution was ultimately preserved at -80 °C.

Exosome characterization

Quantification of exosomal proteins was performed with a BCA assay kit sourced from Sangon Biotech Co., Ltd. (Shanghai, China). The exosome particle characteristics, including diameter and concentration, were assessed through nanoparticle tracking analysis (Flow NanoAnalyzer N30E; NanoFCM Co., Ltd., Fujian, China). Additionally, transmission electron microscopy (H-7650; HITACHI, Tokyo, Japan) revealed a double-layered membrane structure and protein expression was analyzed via western blotting.

Cell culture and treatment

Rat cardiomyocytes H9C2 cells (Oricell Therapeutics Co., Ltd., Shanghai, China) were maintained in Dulbecco's Modified Eagle Medium high glucose (Gibco, Grand Island, NY, United States) supplemented with 10% FBS (Gibco). Cultures were kept at 37 °C under 5% CO₂ in a humidified atmosphere. Once cells reached nearly 80% confluence, they were enzymatically dissociated and subcultured using trypsin-EDTA (Gibco). To induce oxidative stress in H9C2 cells, a modeling concentration of 200 μM H₂O₂ was applied. Cells were pre-incubated with 50 μg/mL exosomes under normoxic conditions for 24 hours, followed by exposure to serum-free medium containing 200 μM H₂O₂ for 12 hours. The following groups of H9C2 cells were established for the in vitro assays: Control group (untreated H9C2 cells), H₂O₂ group (H₂O₂-treated H9C2 cells), H₂O₂ + exosomes group (H₂O₂-treated H9C2 cells following pre-incubation with normoxic exosomes [normo-Exos]), H₂O₂ + hypo-exosomes group (H₂O₂-treated H9C2 cells after pre-incubation with hypoxic exosomes), H₂O₂ + hypo-Exo^antagomir group (H₂O₂-treated H9C2 cells treated with hypoxic exosomes containing miR-486-5p antagomir), and H₂O₂ + hypo-Exo^nc group (H₂O₂-treated H9C2 cells incubated with hypoxic exosomes loaded with a negative control (NC) of miR-486-5p antagomir). All experiments utilized cells between passages 3 and 8.

The internalization of exosomes

To examine the internalization of PKH26-labeled exosomes (Solarbio, Beijing, China), exosomes were initially prepared by diluting them in 250 μL of PBS. This dilution was then combined with an equal volume of 250 μL of Diluent C, followed by the addition of 2 μL of PKH26 that had been pre-diluted in another 250 μL of Diluent C, with an ultimate PKH26 potency of 1 × 10^-6 M. After 5 minutes of incubation, exosome-free FBS was introduced to quench excess dye. The solution underwent ultracentrifugation at 100000 × g for 70 minutes at 4 °C. Exosomes were subsequently harvested and utilized to stimulate H9C2 cells.

Cell viability assay

Following treatment of H9C2 cells, each well received 20 μL of Cell Counting Kit-8 (CCK-8) reagent (Biosharp, Anhui, China) and was maintained at 37 °C for a 2-hour incubation. Absorbance readings were taken at 450 nm, and the result was calculated relative to the control group as a percentage.

Superoxide dismutase assay

According to the instructions, 50 μL of superoxide dismutase (SOD) lysis buffer (Solarbio) was added to the H9C2 cells and kept at 4 °C for 15 minutes. The lysates were then subjected to centrifugation at 12000 rpm for 5 minutes. Supernatants were moved into a 96-well plate and combined with the assay reagent. Following a 30-minute incubation at 37 °C, optical density was measured at 450 nm.

Lactate dehydrogenase leakage assay

Cellular cytotoxicity was evaluated by quantifying extracellular lactate dehydrogenase (LDH) levels using an LDH detection kit (Beyotime, Beijing, China). Supernatants from the culture were harvested and clarified by centrifugation at 1000 rpm for 5 minutes. Subsequently, 60 μL of reaction reagent was dispensed into each well. The absorbance at 490 nm was recorded.

Western blot analysis

Protein samples from cells or exosomes were extracted with RIPA lysis buffer (Solarbio). Concentrations were quantified via the Bradford method. Samples were separated by sodium-dodecyl sulfate gel electrophoresis and subsequently electrotransferred to polyvinylidene fluoride membranes. Rapid Blocking Buffer (Solarbio) was utilized to block the membranes for 15 minutes, followed by the addition of primary antibodies. The membranes were subsequently incubated with secondary antibodies, and the proteins were detected using an enhanced chemiluminescence kit. The intensity of protein bands was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States). Antibodies used for detection included: Calnexin (1:2000; Abcam, Cambridge, United Kingdom), tumor susceptibility gene 101 (1:2000; Proteintech, Wuhan, China), CD63 (1:2000; Abcam), CD9 (1:2000; Proteintech), phosphatase and tensin homolog (PTEN) (1:1000; Abcam), phosphorylated phosphoinositide 3-kinase (p-PI3K) (1:1000; Cell Signaling Technology, Danvers, MA, United States), PI3K (1:1000; Proteintech), p-protein kinase B (p-Akt) (1:1000; Cell Signaling Technology), Akt (1:1000; Proteintech), cleaved caspase 3 (1:1000; Abcam), B-cell lymphoma 2 (Bcl-2) (1:5000; Proteintech), Bcl-2 associated X-protein (BAX) (1:5000; Proteintech), and glyceraldehyde-3-phosphate dehydrogenase (1:8000; Proteintech).

Apoptosis detection with flow cytometry

Cardiomyocyte apoptosis was assessed using flow cytometry in combination with the Annexin V-FITC/PI Apoptosis Kit (Bestbio, Beijing, China). Collected cells were resuspended in binding buffer, followed by the addition of Annexin V-FITC and PI. The proportion of apoptotic cells was determined using the CytoFlex flow cytometer (Beckman Coulter, Brea, CA, United States).

High-throughput miRNA sequencing of exosomes

RNA samples were ligated with 3’ and 5’ adaptors in sequence. The adaptor-ligated RNAs were reverse transcribed to generate cDNA, followed by amplification via polymerase chain reaction. The resulting cDNA libraries underwent gel electrophoresis for size selection to enrich miRNA-sized sequences before sequencing on the platform. Bioinformatics analyses were employed to conduct cluster analysis and evaluate miRNA expression differences between normo-Exos and hypo-Exos. miRNAs were identified based on a significance threshold of P < 0.05, along with predefined criteria for upregulated and downregulated genes. To identify potential target genes for miR-486-5p, the databases TargetScan, microRNA Data Integration Portal, and miRDB were consulted.

Quantitative polymerase chain reaction

TRIzol reagent (Invitrogen, Carlsbad, CA, United States) was utilized to isolate exosomal RNA. Quantitative polymerase chain reaction (qPCR) was conducted utilizing the ALL-in-One miRs RT-qPCR Detection Kit (GeneCopoeia, Rockville, MD, United States). miRNA expression was determined using the 2^-ΔΔCt method, with U6 serving as the internal control. The primer sequences utilized are listed below: For miR-486-5p, the forward primer is 5’-TCCTGTACTGAGCTGCCCCGAG-3’ and the reverse primer is 5’-GATTGAATCGAGCACCAGTTAC-3’; for U6, the forward primer is 5’-CGCTTCGGCAGCACATATACTA-3’ and the reverse primer is 5’-GATTGAATCGAGCACCAGTTAC-3’.

Exo-Fect™ loading of hypo-Exos

Hypo-Exos were subjected to treatment with either the miR-486-5p antagomir or a NC, both sourced from Sangon Biotech using the Exo-Fect™ Exosome Transfection Kit from System Biosciences (Palo Alto, CA, United States). Briefly, purified hypo-Exos were mixed with 20 pmol of RNA oligos and transfection reagents, followed by incubation and precipitation steps. The final exosome pellet was reconstituted in PBS for subsequent applications.

Dual-luciferase reporter assay

Luciferase vectors containing PTEN-wild type (wt) and PTEN-mutant sequences were synthesized by Asia Biotechnology (Shanghai, China). Subsequently, HEK293T cells were transfected with the vectors and either miR-486-5p mimics or NC. Transfection was performed using Lipofectamine 2000 (Invitrogen). miRNA levels were assessed through chemiluminescent analysis with the Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, United States) after adding detection reagents.

Statistical analyses

The experimental results are presented as the mean ± standard error of the mean and percentages. Statistical analyses were conducted on GraphPad 7.0. Student’s t-test and one-way analysis of variance were used to analyze the data. P < 0.05 was considered statistically significant.

RESULTS

Identification of HUC-MSCs and characterization of exosomes

Parental HUC-MSCs were characterized through morphology assessment and flow cytometry analysis. After 3 days of culture under normoxic and hypoxic conditions, HUC-MSCs displayed a clearly defined cytoplasm (Figure 1A), indicating optimal cellular condition and suitability for exosome collection. It is worth noting that hypoxia-preconditioned MSCs exhibited a higher cell density compared with normoxia-treated MSCs at the same time points (Figure 1A), suggesting enhanced proliferative or survival capacity under hypoxic culture conditions. Furthermore, over 95% of cells expressed CD90, CD73, and CD105, with ≤ 5% expressing CD45 and CD34 (Figure 1B). These findings align with the defined phenotypic characteristics of HUC-MSCs. Transmission electron microscopy (Figure 1C) demonstrated the presence of spherical nanoparticles with diameters between 50 nm and 150 nm, and nanoparticle tracking analysis (Figure 1D) displayed a similar size distribution, with mean sizes of 67.45 nm for normo-Exos and 72.83 nm for hypo-Exos. Figure 1E shows the detection of characteristic exosomal markers such as tumor susceptibility gene 101, CD9, and CD63. Conversely, calnexin, an endoplasmic reticulum marker, was present in HUC-MSCs but undetectable in the isolated exosomes (Figure 1E). These complementary methods together confirm that the isolated vesicles are consistent with exosomes.

Figure 1 Identification of human umbilical cord-derived mesenchymal stem cells and characterization of exosomes. A: Representative bright-field microscopy image of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs); B: HUC-MSCs identification by flow cytometry phenotyping; C: Representative electron microscopy image of HUC-MSC-derived exosomes; D: Size distribution of exosomes (Exos) determined by nanoparticle tracking analysis; E: Western blot analysis of the surface markers of HUC-MSCs and exosomes. Hypo-Exos: Hypoxia-preconditioned mesenchymal stem cell-derived exosomes; Normo-Exos: Normoxic exosomes; TSG101: Tumor susceptibility gene 101.

Differential oxygen conditions influence exosome uptake by H9C2 cells

PKH26-stained exosomes were incubated with H9C2 cells for 24 hours. Then fluorescence microscopy was employed to observe exosome uptake. Figure 2A illustrates that H9C2 cells internalized PKH26-labeled exosomes, with the hypo-Exos group exhibiting markedly greater uptake than the normo-Exos group, and Figure 2B demonstrates a statistically significant difference in uptake, indicating that hypo-Exos are internalized more efficiently by H9C2 cells.

Figure 2 Hypoxic preconditioning promoted the internalization of human umbilical cord-derived mesenchymal stem cells exosomes. A: Uptake of the red fluorescence dye PKH26-labeled normoxic exosomes and hypoxia-preconditioned mesenchymal stem cell-derived exosomes (Hypo-Exosome) into H9C2 cells; B: Statistical evaluation of fluorescence intensities. Data are presented as the mean ± standard error of the man (n = 3). ^cP < 0.001, ^dP < 0.0001.

Hypo-Exos ameliorates oxidative stress-induced apoptosis in H9C2 cells

To induce oxidative stress, cardiomyocytes were exposed to H₂O₂ for 12 hours. Cellular viability was evaluated using the CCK-8 assay. Figure 3A shows that the survival rate of H₂O₂-treated cells decreased to approximately 50% of that in the control group. Conversely, the normo-Exos group demonstrated higher viability than the H₂O₂ group. Moreover, treatment with hypo-Exos markedly enhanced the viability of cardiomyocytes vs normo-Exos. The activity of SOD, a key antioxidant enzyme, was also measured. Figure 3B indicates that H₂O₂ resulted in the reduce of SOD activity, and exosome treatment restored SOD activity, with the hypo-Exos group exhibiting the greatest recovery. H₂O₂ induced the elevation of LDH release, an indicator of cellular damage. Exosome treatment, especially hypo-Exos, markedly attenuated LDH release (Figure 3C). Collectively, these findings suggest that hypo-Exos exert superior protective effects in protecting cardiomyocytes from H₂O₂-induced oxidative damage.

Figure 3 Human umbilical cord-derived mesenchymal stem cells exosomes ameliorated oxidative stress-induced apoptosis in H9C2 cells. A: Different effects of normoxic counterparts (normo-Exos) and hypoxia-preconditioned mesenchymal stem cell-derived exosomes (hypo-Exos) on the viability of H9C2 cells; B: Different effects of normo-Exos and hypo-Exos on superoxide dismutase (SOD) activity in hydrogen peroxide (H₂O₂)-induced H9C2 cells; C: Different effects of normo-Exos and hypo-Exos on lactate dehydrogenase (LDH) release in H₂O₂-induced H9C2 cells; D: Expression of apoptosis-related protein was determined by western blotting; E-G: Quantitative analysis of the western blotting; H: Level of apoptosis was determined by flow cytometry; I: Statistical evaluation of flow cytometry. Data are presented as the mean ± standard error of the mean (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001.

Apoptosis is primarily triggered by oxidative stress. We evaluated the expression levels of apoptosis-related proteins to assess the anti-apoptotic potential of hypo-Exos. Figure 3D-G show the upregulation of apoptosis in the H₂O₂-treated group. By contrast, treatment with normo-Exos notably decreased BAX and cleaved caspase-3 levels and enhanced Bcl-2 levels in cardiomyocytes. Moreover, hypo-Exos exerted a stronger modulatory impact on apoptosis-related protein expression than normo-Exos. Additionally, flow cytometry was utilized to assess the protective effect of hypo-Exos on apoptosis. Figure 3H and I show that H₂O₂ markedly induced apoptosis in H9C2 cells; however, treatment with normo-Exos significantly attenuated this effect. Moreover, the hypo-Exos group exhibited a markedly reduced apoptosis rate compared to the normo-Exos group. Collectively, these results suggest that hypo-Exos offer superior protection against apoptosis relative to normo-Exos.

miR-486-5p is involved in the protection of hypo-Exos

We performed miR sequencing to analyze the miRNA differences caused by hypoxic preconditioning. Figure 4A illustrates the supervised clustering for the 25 miRNAs exhibiting the most pronounced abundance variations (P < 0.05) between normo-Exos and hypo-Exos. Of these, 14 miRNAs showed increased expression, while 11 showed decreased expression, as presented in Figure 4B. We chose miR-486-5p as a markedly expressed miRNA, and qPCR further validated a considerable variation in miR-486-5p expression levels (Figure 4C).

Figure 4 miR-486-5p mediated the cell protective effects of hypoxia-preconditioned mesenchymal stem cell-derived exosomes. A: Heat map based on high-throughput microRNAs sequencing representing the 25 identified microRNAs with the most significant abundance differences between normoxic counterparts (normo-Exos) and hypoxia-preconditioned mesenchymal stem cell-derived exosomes (hypo-Exos); B: Volcano plot showing log2 (fold change) on the X-axis and -log10 (P value) on the Y-axis; C: MiR-486-5p expression level in normo-Exos and hypo-Exos determined by quantitative polymerase chain reaction; D: MiR-486-5p antagomir or negative control transfected into hypo-Exos altered the miR-486-5p expression level in hypo-Exos; E: Expression of apoptosis-related protein was determined by western blotting; F-H: Quantitative analysis of the western blotting; I: Level of apoptosis was determined by flow cytometry; J: Statistical evaluation of flow cytometry. All data are representative of three independent experiments and are presented as the mean ± standard error of the mean (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001.

To further validate the contribution of miR-486-5p to the therapeutic action of hypo-Exos, we introduced either the miR-486-5p antagomir or NC into hypo-Exos. qPCR analysis demonstrated a significant suppression of miR-486-5p expression following antagomir transfection (Figure 4D). Moreover, our findings revealed that the antagomir notably weakened the protective capacity of hypo-Exos. Figure 4E-H show that the hypo-Exos-induced downregulation of BAX and cleaved caspase-3 was reversed by miR-486-5p inhibition (comparing H₂O₂ + hypo-Exo^nc with H₂O₂ + hypo-Exo^antagomir), and the upregulation of Bcl-2 induced by hypo-Exos was similarly reversed (comparing H₂O₂ + hypo-Exo^nc with H₂O₂ + hypo-Exo^antagomir). Similarly, flow cytometry results further demonstrated that miR-486-5p antagomir reversed the alleviation of apoptosis rate (Figure 4I and J). By comparison, the incorporation of miR-486-5p NC did not markedly affect the protective effects of hypo-Exos. Collectively, these findings indicate that miR-486-5p loss compromises the anti-apoptotic function of hypo-Exos in H₂O₂-stimulated cardiomyocytes, suggesting the importance of miR-486-5p within hypo-Exos.

Investigation of the signaling mechanism and downstream target associated with the protective effects of exosomal miR-486-5p against apoptosis

To uncover the underlying molecular pathways responsible for the protective function of hypo-Exos, we predicted and analyzed target genes associated with the upregulated miR-486-5p using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. As shown in Figure 5A, KEGG analysis revealed marked enrichment in various signaling pathways. Notably, the PI3K/Akt signaling pathway was particularly prominent. Using three bioinformatics platforms for prediction, we identified PTEN as 1 of 78 common intersections across the platforms (Figure 5B), indicating PTEN as a highly enriched gene in the PI3K/Akt pathway. TargetScan prediction analysis revealed a putative binding region between miR-486-5p and PTEN (Figure 5C). To validate this interaction, luciferase reporter assays were implemented. We observed that co-transfection of PTEN-wt reporters with miR-486-5p mimics led to a notable suppression in luciferase activity (Figure 5D). However, introduction of the mutant 3’ untranslated region constructs did not alter luciferase expression under treatment with miR-486-5p mimic (Figure 5D), confirming the selective nature of this regulatory interaction. These data demonstrate that miR-486-5p acts directly on PTEN.

Figure 5 Potential signaling pathway and target genes involved in the protective effects of exosomal miR-486-5p. A: Kyoto Encyclopedia of Genes and Genomes analyses showed the top 20 significantly enriched signaling pathways of miR-486-5p; B: Venn diagram illustrates the number of targets of miR-486-5p among three bioinformatics platforms; C: MiR-486-5p regulated phosphatase and tensin homolog (PTEN) by directly targeting the 3’ untranslated region (UTR); D: Relative luciferase activity was measured in HEK293T cells transfected with luciferase reporter vectors containing either the wild-type or mutant binding site of miR-486-5p in the 3’ UTR of PTEN, along with a miR-486-5p mimic or negative control (n = 3). ^bP < 0.01.

Hypo-Exos activates PI3K/Akt pathway though miR-486-5p

Subsequently, we investigated the functional status of the PTEN/PI3K/Akt signaling cascade. Figure 6A-D revealed that exposure to H₂O₂ significantly increased PTEN expression while suppressing the PI3K/Akt pathway in cardiomyocytes. By contrast, normo-Exos treatment caused a notable reduction in PTEN levels and enhanced PI3K/Akt pathway activation compared to the H₂O₂ group. Furthermore, hypo-Exos treatment led to a further decrease in PTEN levels and PI3K/Akt pathway activation than normo-Exos treatment. Nevertheless, in the H₂O₂ + hypo-Exo^antagomir group, PTEN protein levels were significantly elevated, and PI3K/Akt pathway activation was decreased compared to the H₂O₂ + hypo-Exo^nc group (Figure 6E-H). Our findings indicate that hypo-Exos downregulate PTEN and stimulate the PI3K/Akt pathway through miR-486-5p.

Figure 6 Exosomal miR-486-5p activated the phosphoinositide 3-kinase/protein kinase B signaling pathway by phosphatase and tensin homolog inhibition. A-H: Expression of phosphatase and tensin homolog (PTEN) and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway protein was determined by western blotting (A and E). Quantitative analysis of the western blotting. All data are representative of three independent experiments and are presented as the mean ± standard error of the mean (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001.

DISCUSSION

Our findings demonstrate that hypo-Exos effectively inhibited apoptosis and oxidative stress induced by H₂O₂. The protective effects of hypo-Exos were achieved through the transfer of miR-486-5p to cardiomyocytes. Specifically, miR-486-5p conferred this effect by directly downregulating PTEN, thereby activating the PI3K/Akt pathway.

Oxidative stress and apoptosis are the common pathophysiological mechanisms of multiple cardiac diseases like heart failure, atrial fibrillation, and myocardial ischemia-reperfusion injury[22-24]. Increased levels of ROS in the body induce oxidative stress, causing abnormal molecular structure and function within cells. For example, ROS triggers mitochondrial DNA damage, thereby causing dysfunction of the respiratory chain complex. Meanwhile, ROS also attacks cell membrane phospholipids, affecting the function of ion channels, and resulting in calcium overload and abnormal electrical activity. In the heart, these pathological changes can promote cardiomyocyte hypertrophy, fibrosis, contractile dysfunction, and impaired cardiac remodeling through the apoptotic pathway[25]. Exosomes represent a potential intervention for cardiac disorders, exerting diverse protective roles including antioxidative, anti-inflammatory, anti-fibrotic, and anti-apoptotic effects. Research has demonstrated that exosomes are highly effective across various cardiovascular conditions, such as heart failure, coronary artery disease, and myocardial ischemia-reperfusion injury[26].

The biological efficacy of exosomes is influenced by the culture conditions of the source cells. MSCs typically reside in a low-oxygen microenvironment in vivo, yet are conventionally cultured under normoxic conditions in vitro. This discrepancy in oxygen tension may alter cellular properties, potentially diminishing the therapeutic potential of exosomes obtained by normoxic culture[27]. Modifying the microenvironment of MSCs has been shown to improve the functional properties of exosomes[28,29]. Recent research has increasingly emphasized the role of hypoxic preconditioning in enhancing exosome activity[30]. Hypoxia enhances MSC stemness, genetic stability, and differentiation potential while boosting proliferation, paracrine signaling, and migration compared to normoxia[29,31,32]. Moreover, exosomes derived from hypoxia-preconditioned MSCs exhibit enhanced cellular uptake efficiency[33]. Hypoxic preconditioning may alter the surface protein and lipid composition of exosomes, such as enrichment in integrins, tetraspanins, or adhesion molecules[34], thereby increasing their affinity for cardiomyocyte surface receptors and facilitating more efficient internalization. Hypoxia-inducible factor 1 alpha (HIF-1a) reportedly transcriptionally regulates miRNA expression under low oxygen conditions, either directly by binding to hypoxia-responsive elements in miRNA promoters or indirectly via modulation of upstream transcription factors[35]. Evidence shows that miR-486-5p expression increases in hypoxic conditions, possibly via HIF-1a-dependent activation[36]. These findings demonstrate that hypoxia preconditioning effectively enhances both exosome yield and therapeutic potency. Lei et al[37] demonstrated that hypoxia-preconditioned exosomes more effectively inhibit hypoxia/reoxygenation-induced ferroptosis and significantly attenuate cardiomyocyte injury. Gao et al’s meta-analysis[38] further confirmed that hypoxia-preconditioned exosomes yield superior cardiac functional recovery in rodent myocardial infarction models. Similarly, our study revealed that hypo-Exos markedly enhanced cell viability, alleviated oxidative damage, and offered superior protection against oxidative stress-induced apoptosis compared to normo-Exos.

Evidence suggests that miR-27b-3p facilitates both cardiomyocyte proliferation and neovascularization, contributing to enhanced cardiac performance[39]. Our study found that hypoxic preconditioning markedly increased the abundance of miR-486-5p within hypo-Exos. Recent research has shown that miR-486-5p mitigates inflammation and prevents apoptosis through suppressing forkhead box protein O1[40]. In addition, miR-486-5p reportedly promotes angiogenesis and supports cardiac functional recovery, thus facilitating myocardial repair[41]. We determined whether miR-486-5p serves as a crucial factor in the cytoprotective actions exerted by hypo-Exos. Notably, suppression of miR-486-5p in hypo-Exos using an antagomir significantly led to pronounced aggravation of apoptosis-related proteins, causing a marked increase in apoptosis. These findings show that miR-486-5p is pivotal in driving the protective effects of hypo-Exos against apoptosis. PTEN, a recognized downstream effector of miR-486-5p[42], acts as a critical inhibitory modulator of the PI3K/Akt pathway[43]. Earlier research has indicated that MSC-derived exosomes can suppress PTEN expression, consequently mitigating apoptosis and promoting survival in cardiomyocytes under hypoxic conditions[44]. By integrating predictions from three bioinformatics platforms with luciferase reporter assays, we further confirmed that miR-486-5p can directly bind to the 3’ untranslated region of PTEN mRNA, resulting in mRNA degradation, thereby decreasing PTEN protein levels.

The PI3K/Akt pathway is commonly recognized because it facilitates cell survival and reduces apoptosis[45]. It is critically involved in controlling cardiomyocyte survival, metabolism, and function within the cardiovascular system[46]. Its activation substantially diminishes cardiomyocyte apoptosis, offering protection under pathological conditions like myocardial infarction, heart failure, and ischemia-reperfusion injury[47]. KEGG pathway analysis in our study highlighted a pronounced overrepresentation of the PI3K/Akt signaling cascade. The modulatory role of exosomes appears to be intimately associated with the PTEN/PI3K/Akt axis[48]. PTEN is a negative regulator of the PI3K/Akt pathway, and its inhibition can lead to phosphatidylinositol-(3,4,5)-trisphosphate accumulation, which recruits and activates Akt via phosphorylation[49]. Additionally, investigations have demonstrated that miR-21 mitigates cardiomyocyte hypoxia/reoxygenation injury and protects cells from oxidative injury via repressing PTEN and modulating the Akt pathway activity[50,51]. Our findings further demonstrated that hypo-Exos-mediated delivery of miR-486-5p significantly downregulates PTEN expression, which subsequently triggers activation of the PI3K/Akt signaling cascade in cardiomyocytes.

This study adds to the growing evidence supporting hypo-Exos as promising therapeutic candidates for cardiovascular diseases, particularly those linked to oxidative stress. Our results show that hypoxic preconditioning boosts the cardioprotective efficacy of MSC-derived exosomes and underscores the importance of miR-486-5p in mediating these effects. Hypo-Exos enhance antioxidant defenses and regulate apoptotic pathways, providing a more effective therapeutic strategy than conventional MSC-derived exosomes. This manuscript’s key contribution is the detailed elucidation of the molecular mechanisms mediating the protective effects of hypo-Exos, specifically the involvement of miR-486-5p and the PTEN/PI3K/Akt pathway (Figure 7). This work offers a promising approach for targeted therapies addressing oxidative stress-related cardiovascular conditions.

Figure 7 Hypoxic preconditioned human umbilical cord-derived mesenchymal stem cells derived exosomes alleviate H9C2 cells apoptosis and oxidative stress through the miR-486-5p targeting phosphoinositide 3-kinase/protein kinase B pathway. Akt: Protein kinase B; HUC-MSCs: Human umbilical cord-derived mesenchymal stems; PI3K: Phosphoinositide 3-kinase; PTEN: Phosphatase and tensin homolog.

This study had several limitations. First, our analysis focused solely on alterations in miRNA content, without addressing other exosomal components. The potential roles of these additional RNAs and proteins in hypo-Exos necessitate further exploration. Furthermore, miR-486-5p in hypo-Exos may not be the only miRNAs responsible for oxidative stress-related damage in H9C2 cells. Other miRNAs in hypo-Exos may also contribute to cardiomyocyte survival. While miRNAs mediate multiple targets and pathways, our study identifies PTEN as a potential target. Future research should investigate other genes involved in cardioprotection. Finally, miR-486-5p may have additional downstream targets that contribute to its cardioprotective effects, which require further investigation.

CONCLUSION

In summary, hypo-Exos treatment enhances cell survival, alleviates oxidative stress injury, and reduces apoptosis through the delivery of miR-486-5p in H₂O₂-treated cardiomyocytes. The underlying mechanism involves miR-486-5p’s direct inhibition of PTEN, thereby modulating the PI3K/Akt signaling pathway.

ACKNOWLEDGEMENTS

The authors express their sincere gratitude to all the staff who contributed to this project for their efforts.