Exosomal miR-218-5p/miR-363-3p from Endothelial Progenitor Cells Ameliorate Myocardial Infarction by Targeting the p53/JMY Signaling Pathway

Accumulating evidence has shown that endothelial progenitor cell-derived exosomes (EPC-Exos) can ameliorate myocardial fibrosis. The purpose of the present study was to investigate the effects of EPC-Exos-derived microRNAs (miRNAs) on myocardial infarction (MI). A miRNA-Seq dataset of miRNAs differentially expressed between EPCs and exosomes was collected. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the miRNA expression indicated by miRNA-Seq. Immunofluorescence, cell proliferation, and angiogenesis assays were employed to investigate the effects of miRNAs on cardiac fibroblasts (CFs) in vitro. Interactions between miRNAs and their respective targets were examined via immunoblotting, qRT-PCR, and luciferase reporter assays. An MI rat model was constructed, and various staining and immunohistochemical assays were performed to explore the mechanisms underlying the miRNA-mediated effects on MI. miR-363-3p and miR-218-5p were enriched in EPC-Exos, and miR-218-5p and miR-363-3p mimic or inhibitor enhanced or suppressed CF proliferation and angiogenesis, respectively. miR-218-5p and miR-363-3p regulated p53 and junction-mediating and regulatory protein (JMY) by binding to the promoter region of p53 and the 3′ untranslated region of JMY. Additionally, treatment of CFs with Exo-miR-218-5p or Exo-miR-363-3p upregulated p53 and downregulated JMY expression, promoted mesenchymal-endothelial transition, and inhibited myocardial fibrosis. Administration of exosomes containing miR-218-5p mimic or miR-363-3p mimic ameliorated left coronary artery ligation-induced MI and restored myocardial tissue integrity in the MI model rats. In summary, these results show that the protective ability of EPC-Exos against MI was mediated by the shuttled miR-218-5p or miR-363-3p via targeting of the p53/JMY signaling pathway.


Introduction
Cardiovascular diseases account for over 40% of deaths annually and are the leading cause of death around the world [1]. Of note, chronic heart failure (CHF) is the leading cause of cardiovascular mortality, with an increased prevalence in recent years [2]. Myocardial infarction-(MI-) induced remodeling of the left ventricle (LV) is the most common cause of CHF [3]. The clinical features of MI include LV dilatation, cardiomyocyte death, and myocardial remodeling [3,4]. Irreversible cardiac tissue damage caused by MI leads to myocardial fibrosis (MF), ventricular remodeling, cardiac dysfunction [5], and finally heart failure [6][7][8]. MF is the final pathological consequence of cardiovascular disease and is characterized by abnormal thickening of heart valves or excessive deposition of extracellular matrix in the cardiac muscle [9], which results in progression to heart failure [10,11]. Therefore, alleviation or prevention of MF is an important therapeutic strategy for the management of MI.
Endothelial progenitor cells (EPCs) originate from the bone marrow, localize within damaged tissues, and play a vital role in the regeneration of the endothelial lining of damaged blood vessels during heart attacks [12], representing a possible therapeutic treatment for MI. Previous studies have reported that a higher level of circulating EPCs predicted a better outcome for patients with cardiovascular disease [13]. Smallscale clinical trials have confirmed EPCs as a potential treatment for MF [14] and MI [15,16]. However, to date, the functional substrates and potential molecular mechanisms associated with EPC therapy for MF or MI have not been thoroughly elucidated. Previous studies have demonstrated that CFs have the potential to transform into pluripotent stem cells, myoblasts, neurons, and endothelial cells [17]. More importantly, exosomes from EPCs have the potential to promote CFs differentiated into endothelial cells by upregulating the expression of mesenchymal-endothelial transition-(MEndT-) related genes and increasing the expression of high-mobility group box 1 protein B1 [18]. However, the regulatory mechanisms underlying the role of CFs in MI remain elusive.
Exosomes are secreted by various cell types [19,20] and contain various paracrine factors, including proteins, lipids, and RNA. Moreover, exosomes vary in diameter from 40 to 100 nm [21] and can be used to mediate intercellular communication [22]. EPC-derived exosomes (EPC-Exos) can accelerate cutaneous wound healing [23] and ameliorate acute lung injury [24], opening a new horizon for therapeutic applications of EPC-Exos. Our previous study confirmed that EPC-Exos induce proliferation and angiogenesis of CFs and provide promising therapeutic effects for MF [18].
Emerging evidence indicates that exosomes can transport functional microRNAs (miRNAs) to induce degradation of target gene messenger RNA (mRNA) [25,26] and play significant roles in physiology and disease [27,28]. Circulating miRNAs can also serve as putative biomarkers of MF [29]. miRNA-enriched exosomes contribute to cardiac nuclear factor (erythroid-derived 2)-like 2 (Nrf2) dysregulation and have the potential to reduce MI-related adverse events and attenuate MI-induced myocardial injury [30]. Therefore, we hypothesized that the therapeutic effects of EPC-Exos on MF or MI may be mediated by miRNAs released from EPC-Exos.
To date, there is no reported research on the therapeutic potential of miRNAs secreted by EPC-Exos for MI. In the present study, we characterized the functional miRNAs secreted from EPC-Exos and confirmed the effects of these miRNAs on the proliferation and angiogenesis potential of CFs. In addition, the biological roles of functional miRNAs were evaluated in experimental rats with MI established via left coronary artery (LCA) ligation.

Sequence Analysis of EPC-Exos miRNAs. Total RNA from
EPCs and EPC-Exos was isolated by means of a mirVana miRNA isolation kit (Ambion, Austin, TX, USA). One biological repeat with three mixed samples for EPCs and EPC-Exos was used for the sequence analysis. The concentration and quality of RNA samples were detected with an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA), and 50 ng RNA samples were fragmented and used for miRNA sequencing on Genome Analyzer IIx (Illumina, San Diego, CA, USA). After generating the sequences, a BLASTN search was utilized to identify conserved miRNAs, and Rfam was employed (European Molecular Biology Laboratory, Heidelberg, Germany) for the deletion of non-miRNA sequences. miRDeep2 was used to predict novel miRNAs. The length distribution of the cells and exosomal miRNAs was analyzed, and miRNAs with differential expression between cells and exosomes were detected as described previously [31]. Gene Ontology (GO) categories [32] and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses [33] were utilized to investigate specific functional roles of differentially expressed miRNAs. GO enrichment analysis, including biological processes, cellular component, and molecular function, identified which GO terms were over or underrepresented within a given set of genes. The KEGG knowledge database, an integrated database resource, is generally used to identify functional and metabolic pathways. To determine whether the expression changes in miR-218-5p and miR-363-3p were influenced by the number of exosomes secreted from EPCs, GW4869 (an inhibitor of neutral sphingomyelinase 2, 10 μM for 12 h, MCE China) was used to treat EPCs, and then, EPC-Exos were isolated using a Total Exosome Isolation Reagent Kit (4478359, Thermo Fisher, USA).

Quantitative Real-Time Reverse Transcription-Polymerase
Chain Reaction (qRT-PCR). Total RNA samples were extracted from EPCs, EPCs-Exos, and tissues around an infarct using Trizol reagent (Takara, Dalian, China). The synthesis of cDNA used for gene and miRNAs was performed using a Bestar™ qPCR RT kit (DBI; #DBI-0) and Bestar™ miRNA qPCR RT kit (DBI; #2220), respectively. The relative mRNA levels of α-SMA, vimentin, CD31, VEGFR-2p53, JMY, and miRNAs were determined via real-time quantitative PCR using a 20 μL reaction system. PCR was performed on an ABI PRISM 7300 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) using the following conditions: 95°C for 2 min, followed by 40 cycles of 94°C for 20 sec, 58°C for 20 sec, and 72°C for 20 sec. GAPDH or U6 was used as the internal reference for genes and miRNAs, respectively. Fold changes were determined via 2 −ΔΔCt (where ΔCt = ðCt of miRNA of interestÞ − ðCt of U6Þ, and ΔΔCt = ðΔCt of miRNA of interestÞ − ðΔCt of U6Þ) for at least three biological repeats with three technological replicates. All the primer sequences used are listed in Supplemental Table 1 Probes) and incubated with the cells for 1 h at room temperature. The nuclei were stained with Hoechst 33258. Immunostaining was visualized with a fluorescence microscope (Olympus inverted microscope IX71) and quantitated using flow cytometry. Cells within the field of view under the same magnification and with the same cell seeding density before the experiment were analyzed in the study.
For detection of tube-like structure formation, Matrigel (300 μL) was plated into the bottom of 6-well plates at 37°C for 30 min, and 10,000 CFs were seeded on the Matrigel and treated with exosomes containing miRNAs for 24 h. An inverted microscope (Olympus) was used to assess tube formation.
2.9. Animal Experimental Model. Three-month-old healthy male SD (Sprague-Dawley) rats (230-250 g) were used for experimentation in accordance with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 8th Edition, 2011). The protocol was approved by the Committee for the Ethics of Animal Experiments of the First Affiliated Hospital of Sun Yat-sen University. The rats were fixed on a board and anaesthetized via intraperitoneal administration of 100 mg/kg ketamine + 10 mg/kg xylazine. Surgery was performed under sterile conditions. We disinfected the chest of the rats and lightly pressed on the right side of the thorax, extruding the heart and insert-ing a needle 2 mm below the root of the left atrial appendage to a needle depth of 0.5 mm. We threaded the surface of the myocardium near the pulmonary arterial cone with a 6/0 suture, rapidly ligated the left anterior descending artery, closed the thorax, and finally sutured the incisions layer by layer. The rats were allowed to move freely after surgery and were injected with 400,000 U penicillin to prevent infection. Postoperative medication (subcutaneous injection of 0.05 mg/kg buprenorphine) at 12 h and 24 h after surgery was also used to alleviate any discomfort.
After the operation, when the animals recovered spontaneous breathing, rats whose electrocardiogram (ECG) showed significantly increased J points and/or tall and biphasic or inverted T waves and/or Q waves were considered to be MI rats. The animals that met the above ECG requirements were randomly divided into three groups: MI group (model), MI with exosomes containing miR-218-5p mimic group, and MI with exosomes containing miR-363-3p mimic group, with ten rats in each group. Pooled exosomes (300 μg) derived from EPCs with miR-218-5p mimic or miR-363-3p mimic transfection were first resuspended in 150 μL of PBS [36,37] and injected intramyocardially with an insulin syringe through a 21-gauge needle into the border of the visually recognizable ischemic area at 24 h after surgery. The sham-operated group without any surgery and the control model rats were injected with 150 μL of PBS. After eight weeks, echocardiography was used to detect cardiac function in the rats. Capillary density was observed and calculated under an optical microscope.

Histological Evaluation.
The heart tissues of rats in the different groups were immediately dissected, fixed in 10% buffered formalin, and processed in a paraffin tissue processing machine. Seven-micrometer sections were stained with hematoxylin and eosin (H&E, G1005, Servicebio, China), Masson's trichrome (G1006, Servicebio, China), and Van Gieson (G1046, Servicebio, China), and the tissues were assessed. Representative photomicrographs were observed under a microscope. For measurement of infarct volumes, the excised left ventricles of the hearts were frozen and sectioned from the apex to the base into 3 mm slices. The slices were immersed in 2% TTC (2,3,5-triphenyltetrazolium chloride, T8877, Sigma-Aldrich) solution in PBS for 30 min at 37°C in the dark. The slices were fixed in 10% formaldehyde, and the area of the ischemic damage was measured using a morphometric program (Digi Cell 4.0).
2.11. Immunohistochemistry. Heart tissue slices were blocked by incubation with 3% H 2 O 2 after deparaffinization and rehydration and washed with 0.05 M ethylenediaminetetraacetic acid (EDTA) followed by 4% paraformaldehyde. The tissues were incubated in 5% dry milk and 0.5% goat serum for 20 min. Then, 5 μm sections were incubated with anti-von Willebrand factor (anti-vWF) antibody in the presence of 10% rabbit serum overnight at room temperature. The sections were incubated with horseradish peroxidase-(HRP-) conjugated goat anti-rabbit IgG secondary antibody for another two hours. Diaminobenzidine (DAB) staining (K5007, Dako) was used for examination. The slides were counterstained with Harris hematoxylin to stain cell nuclei. The heart tissue slices 4 Oxidative Medicine and Cellular Longevity were also incubated with anti-α-SMA antibody and the respective secondary antibody. The slides were counterstained with DAPI to stain cell nuclei.

Statistical Analysis.
All the data are presented as the means ± standard deviation (SD). Each in vitro experiment was performed in triplicate. All the data produced by qRT-PCR were based on at least 3 biological repeats with at least 3 technical repeats. Statistical significance between/among different treatment groups was calculated using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test or an unpaired Student t-test, as appropriate, using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). P < 0:05 was considered to be statistically significant.

Overview of Small RNA EPC and EPC-Exos Sequencing Data.
A previous study demonstrated that EPC-Exos play a vital role in CF proliferation and angiogenesis [18]. To investigate similarities or differences in specific patterns of miRNA expression between cells and exosomes, we first isolated exosomes and identified them via transmission electron microscopy (Supplemental Figure 1A), particle diameter size distribution (Supplemental Figure 1B and Source data), and western blot analysis (Supplemental Figure 1C). The distribution of exosomes in EPCs was assessed by PKH67 staining (Supplemental Figure 1D). In an initial effort to identify differentially expressed miRNAs in exosomes that could account for their functions relative to EPCs, we profiled the expression of miRNAs using DESeq2. A total of 7,342,859 and 8,898,449 clean reads were obtained with 6,997,238 and 8,813,698 adapter-trimmed reads (length ≥ 15 nucleotides) from EPCs and exosomes, respectively (Supplemental Table 5). Further analysis showed that the number of reads aligned to known human pre-miRNAs in miRbase21 was 4,691,984 and 127,888 in EPCs and exosomes, respectively (Supplemental Table 5. Unique small RNAs with lengths ranging from 16 to 30 nucleotides were obtained by filtering the low-quality reads or sequence reads with a length less than 15 nucleotides, and the length distribution of unique small RNA sequences (17-30 nucleotides) in the cells and exosomes was similar (Supplemental Figure 2A). The lengths of unique small RNAs in the cells and exosomes ranged between 20 and 24 nucleotides, with the most abundant size class being 22 nucleotides followed by 23 and 21 nucleotides (Supplemental Figure 2A). The new miRNAs were discovered using miRDeep2 [38]. Reads with a removed adaptor, sequence length ≥ 17 base pairs, and mismatch number ≤ 1 were used to perform novel miRNA prediction, and 144 novel miRNA candidates were detected in chromosomes of both cell types and exosomes (Supplemental Figure 2B and Supplemental Table 6).
3.3. miR-218-5p and miR-363-3p Promote CF Angiogenesis and Proliferation. To investigate the functional potential of miR-NAs in MF, three miRNAs, namely, miR-6087, miR-218-5p, and miR-363-3p, which showed the most significant changes in the expression profiles, were selected for further examination. EPCs were transfected with mimics and inhibitors of the miRNAs, and their expression levels were detected in EPC-Exos using qRT-PCR (Figure 2(a)). The expression of miR-218-5p and miR-363-3p was significantly upregulated with miRNA mimic transfection and downregulated with miRNA inhibitor transfection in EPC-Exos compared with the NC-Exo group (Figure 2(a)). However, the expression of miR-6087 was not affected (Figure 2(a)). To determine whether the expression changes in miR-218-5p and miR-363-3p were caused by the number of exosomes secreted from EPCs, GW4869 (an inhibitor of neutral sphingomyelinase 2) was used to treat EPCs. As shown in Supplemental Figure 4A and 4B, the concentration of exosomes in EPCs treated with GW4869 was significantly decreased compared with the control. Further analysis showed that the expression of miR-218-5p and miR-363-3p was significantly increased in cells treated with GW4869 compared with control cells but significantly decreased in EPC-Exos (Supplemental Figure 4C and 4D). Therefore, miR-218-5p and miR-363-3p were selected for further experiments. To investigate whether the two candidate miRNAs are involved in the mediation of CF proliferation and    7 Oxidative Medicine and Cellular Longevity angiogenesis, CFs (NC-Exo), CFs+EPC-Exos with miR-218-5p mimic (mimic-218-Exo), CFs+EPC-Exos with miR-218-5p inhibitor (inhibitor-218-Exo), CFs+EPC-Exos with miR-363-3p mimic (mimic-363-Exo), and CFs+EPC-Exos with miR-363-3p inhibitor (inhibitor-363-Exo) were established. The number of capillary-like structures was increased in the mimic-218-Exo and mimic-363-Exo groups compared with the NC-Exo group and was decreased in the inhibitor-218-Exo and inhibitor-363-Exo groups (Figure 2(b)). Further analysis showed that the tube length in CFs with miRNA mimic-Exo transfection was significantly longer than that in the control, while shorter tube lengths were observed in the miRNA inhibitor-Exo groups (Figure 2(b)). These results indicate that miR-218-5p and miR-363-3p can promote angiogenesis of CFs. CCK8 assays demonstrated that treatment with miR-218-5p and miR-363-3p mimic-Exo dramatically increased cell viability compared to the control, while miRNA inhibitor-Exo significantly decreased cell viability at 48 h and 72 h (Figure 2(c)). These findings suggest that miR-218-5p and miR-363-3p can promote CF proliferation. To verify the results of this experiment, we performed a BrdU labelling assay. The results confirmed that EPC-Exos with miRNA mimic significantly promoted the proliferation of CFs, because the number of BrdU-positive cells found in CFs treated with mimics was greater than that in the inhibitor and control groups (Figure 2(d)). Therefore, miR-218-5p and miR-363-3p promoted CF proliferation.
In order to further confirm that miR-218-5p and miR-363-3p play important roles in CF proliferation and angiogenesis related to EPC-Exos, the function analysis of EPC-Exos to CF proliferation and angiogenesis was performed in a coculture system. CCK8 assays showed that cell viability was dramatically increased in the Exo group compared with control while significantly decreased when treated with GW4869 compared with the Exo group at 48 h (Supplemental Figure 5A). Further analysis showed that the number of capillary-like structures and the tube length in CFs was increased in the Exo group compared with control while decreased when treated with GW4869 compared with the Exo group (Supplemental Figure 5B). These results demonstrated that miR-218-5p and miR-363-3p which promoted CF proliferation and angiogenesis were from EPC-Exos.
To further confirm the changes in CF angiogenesis and proliferation caused by miR-218-5p and miR-363-3p, CFs were directly transfected with miR-218-5p and miR-363-3p.  Oxidative Medicine and Cellular Longevity miR-218-5p and miR-363-3p were successfully overexpressed or suppressed in CFs harboring mimic or inhibitor, respectively (Supplemental Figure 6A). Further analysis showed that the expression of p53 was significantly upregulated in the mimic-218 (miR-218-5p mimic) group and downregulated in the inhibitor-218 (miR-218-5p inhibitor) group compared with the NC group. In addition, the expression of JMY was significantly downregulated in the mimic-363 (miR-363-3p mimic) group but upregulated in the inhibitor-363 (miR-363-3p inhibitor) group compared with the NC group (Supplemental Figure 6B). In addition, the results showed that tube length was significantly elevated in both the mimic-218 and mimic-363 groups and decreased in the inhibitor group compared with the NC group (Supplemental Figure 6C and 6D). BrdU labelling showed that the number of BrdU-positive cells found in CFs treated with mimic-218 or mimic-363 was more than that in the control groups, while the number in the inhibitor-treated group was decreased (Supplemental Figure 6E and 6F).
3.4. miR-218-5p and miR-363-3p Promoted Mesenchymal-Endothelial Transition in CFs. In our previous study, we demonstrated that EPC-Exos promoted MEndT to reduce MF [18]. To test the functional potential of miR-218-5p and miR-363-3p in MEndT, we detected the specific endothelial lineage characteristics of CFs using a phagocytosis assay. The results showed that phagocytosis in CFs dramatically increased in the mimic-218-Exo group (Figure 3(a)) and the mimic-363-Exo group (Figure 3 3.5. p53/JMY Were Respective Targets of miR-218-5p and miR-363-3p. To investigate the molecular mechanism of the miR-218-5p-and miR-363-3p-mediated effects on MF, we utilized TargetScan to identify the potential binding sites of miR-218-5p and miR-363-3p. Systematic bioinformatics analysis revealed that miR-218-5p could bind with the 5 ′ -AAGCAC-3 ′ site in the promoter region of p53 and that miR-363-3p could bind with the 5 ′ -GUGCAAU-3 ′ site in the 3 ′ UTR of JMY mRNA at two sites (Figures 6(a) and 6(d)). Therefore, a luciferase reporter assay was used to identify interactions between miR-218-5p and the p53 promoter, and miR-363-3p and the 3 ′ UTR of JMY. No obvious changes were observed in the NC and cel-miR-39 mimic groups, which harbored the wild-type and mutant p53 promoter. However, luciferase activity was significantly upregulated after cotransfection with miR-218-5p mimic and the reporter vector with the wild-type p53 promoter, while no significant change was observed with the mutant p53 promoter (Figures 6(b) and 6(c)). In addition, no obvious changes were found in the NC and cel-miR-39 mimic groups, harboring the wild-type and mutant JMY 3′UTR, regardless of whether the P1 or P2 binding site was present. However, luciferase activity was significantly decreased after cotransfection with miR-363-3p mimic and the reporter vector with wild-type JMY at the P2 binding site, while no significant change was observed for the P1 binding site (Figures 6(e) and 6(f)). Therefore, miR-218-5p could bind the p53 promoter region, and miR-363-3p could bind P2 sites in the 3 ′ UTR of JMY. Furthermore, the mRNA expression of TP53 (P53) was upregulated in the mimic-218-Exo group and downregulated in the inhibitor-Exo group compared to the NC-Exo group ( Figure 6(g)). Moreover, the mRNA expression of JMY was significantly downregulated in the mimic-363-Exo group and upregulated in the inhibitor-Exo group compared with the NC-Exo group (Figure 6(g)). The expression changes in TP53 and JMY were further verified by immunofluorescence staining (Figures 7(a) and 7(c)) and western blot analysis (Figures 7(b) and 7(d)). In addition, the expression of p53 was suppressed and JMY was overexpressed in CFs (Figure 7(e)) compared with the mimic-218-Exo and mimic-363-Exo groups, respectively. We also found that both tube length (Figure 7(f)) and the number of BrdU-positive cells (Figure 7(g)) were dramatically decreased in both the si-p53 and OE-JMY groups compared with the mimic-218-Exo and mimic-363-Exo groups, respectively. In general, our results indicate that EPC-Exos regulate the proliferation, angiogenesis, and tube formation of CFs through miR-218-5p-and miR-363-3p-mediated p53/JMY signaling pathways.
3.6. miR-218-5p and miR-363-3p Mimic Alleviated LCA Ligation-Induced MI. To investigate whether exosomal miR-218-5p and miR-363-3p can ameliorate LCA ligationinduced MI in vivo, we first constructed a MI rat model by injecting EPC-Exos containing miR-218-5p and miR-363-3p mimic. ECGs showed inverted QRS waves in the MI model group (model) compared to the sham-operated group (sham), suggesting successful establishment of an MI model (Figure 8(a)). Injection of EPC-Exos with miR-218-5p and miR-363-3p mimic attenuated the impaired cardiac function, indicating a therapeutic effect on MI (Figure 8(a)). Furthermore, LVEDd, LVESd, and LVIDs were increased in the model group, and these effects were partially reversed by treatment with EPC-Exos containing miR-218-5p and miR-363-3p mimic (Supplemental Table 4). Similarly, the decreased parameter values in the model rats, including HR, LVIDd, SV, LVEF, FS, CO, LVPWT, and LVAWT, were also partially restored to the values seen in sham rats       Figure 5: Western blot analysis to detect the relative expression levels of CD31, VEGFR-2, α-SMA, and vimentin in CFs treated with miR-218-5p (a) and miR-363-3p (b) mimic-Exo and inhibitor-Exo. The data are presented as the mean ± SD (n = 3). * P < 0:05, * * P < 0:01, and * * * P < 0:001, mimic-Exo or inhibitor-Exo versus NC-Exo. 12 Oxidative Medicine and Cellular Longevity   14 Oxidative Medicine and Cellular Longevity after treatment with EPC-Exos containing miR-218-5p and miR-363-3p mimic (Supplemental Table 4). For TTC staining, a large core volume of tissue injury was found in the model group (Figure 8(b)). However, the core volume steadily decreased to approximately 50% in the mimic-218-Exo and mimic-363-Exo groups compared with the model group (Figure 8(b)). Subsequently, significant increased expression of miR-218 and miR-363 was observed in both the mimic-363-Exo and mimic-218-Exo groups compared with the sham and model groups (Figure 8(c)). Furthermore, we found that the expression of JMY was significantly decreased in the mimic-363-Exo group while the expression of p53 was significantly elevated in the mimic-218-Exo group compared with the model group (Figures 8(d) and  8(e)). Western blotting also confirmed these results ( Figure 8(f)). In addition, we detected the expression of collagen-1, collagen-3, Timp-1, Timp-2, Timp-3, and Timp-4. As shown in Figure 8(f), the expression levels of collagen-1 and collagen-3 were significantly decreased, while those of Timp-1 and Timp-3 were upregulated (no change in the expression of Timp-2 and Timp-4) in both the mimic-218-Exo and mimic-363-Exo groups compared with the model group (Figure 8(f)). Therefore, exosomal miR-218-5p and miR-363-3p can reduce cardiac dysfunction in MI rats. Histological examination of the rat heart tissues was further performed. As shown in Figure 9(a), after the challenge of the LCA ligation, H&E staining of heart tissues showed typical MI pathological traits. However, these histopathological changes were clearly attenuated in the mimic-218-Exo and mimic-363-Exo groups, reflected by visible  Figure 7: Effect of miR-218-5p and miR-363-3p on p53/JMY. (a, c) Immunofluorescence staining for the relative expression levels of p53 and JMY in CFs, in the miR-218-5p/miR-363-3p mimic-Exo and inhibitor-Exo groups. (b, d) Western blot analysis to detect the relative expression levels of p53 and JMY in miR-218-5p/miR-363-3p mimic-Exo and inhibitor-Exo groups in CFs, respectively. The photographs were taken with a laser confocal microscope (Zeiss, LSM700), magnification: 120x. The data are presented as the mean ± SD (n = 3). * P < 0:05 and * * P < 0:01, mimic-Exo or inhibitor-Exo versus NC-Exo. (e) Western blot analysis to detect the relative expression levels of p53 and JMY in si-p53 and OE-JMY CF groups, respectively. The data are presented as the mean ± SD (n = 3). * * P < 0:01, si-p53 or OE-JMY group versus mimic-218-Exo or mimic-363-Exo group. (f) The tube formation capability and relative tube length were decreased in the si-p53 and OE-JMY groups. The data are presented as the mean ± SD (n = 3). * * P < 0:01, si-p53 or OE-JMY group versus mimic-218-Exo or mimic-363-Exo group. (g) BrdU labelling and flow cytometry assay to detect the effect of si-p53 and OE-JMY on cell proliferation. The data are presented as the mean ± SD (n = 3). * * P < 0:01, si-p53 or OE-JMY group versus mimic-218-Exo or mimic-363-Exo group. 15 Oxidative Medicine and Cellular Longevity

16
Oxidative Medicine and Cellular Longevity fibers with cross-linked-shaped striated muscle tissue containing nuclei located on the periphery of the cell and remodeling of the intercalated disc connections (Figure 9(a)). Masson and Van Gieson staining confirmed a reduced degree of cardiac fibrosis with downregulation of collagen expression in the mimic-218-Exo and mimic-363-Exo groups (Figure 9(a)). Moreover, the expression of α-SMA was dramatically reduced in the mimic-218-Exo and mimic-363-Exo groups compared to the model group (Figure 9(b)). Furthermore, immunohistochemistry assays showed that the von Willebrand factor (vWF) expression level was partially restored in the mimic-218-Exo and mimic-363-Exo groups compared to the model group (Figure 9(c)). As a risk factor for patients with MI, vWF plays a vital role in hemostasis and is always elevated in the MI rat model [39]. We also quantified capillary density.
In line with the histological examinations, the number of vessels/field area in the remodeled left ventricle in the mimic-218-Exo and mimic-363-Exo groups was increased compared with that in the model group (Figure 9(d)). These findings confirm that exosomal miR-218-5p and miR-363-3p can alleviate LCA ligation-induced MI.

Discussion
Cardiovascular disease not only places a severe burden on affected patients and their families but also influences society and economic development, underscoring the need for innovative new therapies [40]. Myocardial infarction-induced remodeling of the left ventricle is the most common cause of CHF, which is the leading cause of cardiovascular mortality [1]. Therefore, exploring the regulation mechanisms underlying MI is of great significance for control of cardiovascular disease development. Accumulating evidence has shown that exosomes can play a role in therapeutic effects in MI and that miRNA plays an important role in MI [41][42][43]. However, the molecular mechanism of EPC-Exos miRNAs in MI is rarely studied. Here, we examined the role of exosome-derived miRNA in regulating angiogenesis, proliferation, and mesenchymal-endothelial transition of CFs during MI in vitro and in vivo. The results showed that exosomal miR-218-5p and miR-363-3p from endothelial progenitor cells promoted angiogenesis, proliferation, and mesenchymalendothelial transition of CFs by targeting the p53 promoter and JMY 3 ′ UTR, respectively, which further alleviated LCAinduced chronic myocardial infarction. Our study will shed new light on Exo-miRNA-based therapy for MI. MicroRNAs (miRNAs) are small noncoding RNAs that block translation or induce degradation of mRNA and thereby control patterns of gene expression. Increasing studies have found that miRNAs play crucial roles in MI [44][45][46]. For example, microRNA-133 overexpression promotes the therapeutic efficacy of mesenchymal stem cells in acute myocardial infarction [47]; Li et al. demonstrated that intravenous miR-144 reduces left ventricular remodeling after myocardial infarction [48]. miR-208, miR-494, miR-499, and miR-1303 expression levels are considered to be biomarkers in the early diagnosis of acute myocardial infarction [49]. MicroRNA-21 was fund to mediate the protective effect of cardiomyocyte-derived conditioned medium on ameliorating myocardial infarction in rats [50]. These results demonstrate that different miRNAs play important roles in MI. As small nanometer-sized vesicles and intercellular shuttles, exosomes essentially transfer loading proteins and RNAs for offloading in target cells and modulate gene and protein expression, thereby regulating cell activity [42,51]. And Luo et al. revealed that exosomes from miR-126-overexpressing ADSCS were therapeutic in relieving acute myocardial ischemic injury [52]. Tian   prevention of cell death in myocardial infarction [53].
Recently, it showed that serum exosomal miR-21, miR-126, and PTEN are novel biomarkers for diagnosis of acute coronary syndrome [54]. These results demonstrate that exosomal miRNA plays key roles in MI and might provide a novel therapeutic pathway for relieving MI. In the present study, 385 differentially expressed miRNAs, with 100 upregulated and 285 downregulated, were found in EPC-Exos compared with EPCs

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Oxidative Medicine and Cellular Longevity by sequencing and profiling miRNA expression in EPC-Exos. miR-218-5p and miR-363-3p showed the most significant changes in the expression profiles, demonstrating the important roles of miR-218-5p and miR-363-3p in MI. Several miRNAs have been shown to control important processes that contribute to the pathophysiological consequences of MI, such as regulating cardiomyocyte cell death and postischemic neovascularization, controlling cardiomyocyte proliferation, or interfering with cardioprotective effects mediated by stem or progenitor cells [55][56][57]. In addition, miRNAs can be used for direct reprogramming of cardiac fibroblasts into cardiomyocytes [41]. A recent study showed that EPC-Exos contribute to CF proliferation and angiogenesis and ameliorate MF [18]. Arif et al. revealed that microRNA-210-mediated proliferation, survival, and angiogenesis promoted cardiac repair postmyocardial infarction in rodents [58]. Cardiomyocyte-derived exosomal microRNA-92a mediated postischemic myofibroblast activation both in vitro and ex vivo [59]. In addition, Fan et al. demonstrated that microRNA-210 promotes angiogenesis in acute myocardial infarction [60]; M1-like macrophage-derived exosomes suppressed angiogenesis and exacerbate cardiac dysfunction in a myocardial infarction microenvironment [61]. Ma et al. revealed that microRNA-132 delivered by mesenchymal stem cell-derived exosomes can promote angiogenesis in MI [62]. Therefore, we speculated that miR-218-5p and miR-363-3p might be involved in CF proliferation and angiogenesis in MI. Consistent with this hypothesis, our study revealed that miR-218-5p and miR-363-3p mimics promoted angiogenesis and proliferation in CFs. In our previous study, we demonstrated that EPC-Exos promoted MEndT to reduce MF [18]. In addition, Bayoumi et al. demonstrated that microRNA-532 protects the heart in acute myocardial infarction via endothelial-to-mesenchymal transition by suppressing prss23 [63]. Our in vivo and in vitro experiments showed that miR-218-5p and miR-363-3p induced MEndT of CFs via upregulation of endothelial cell markers (CD31 and VEGFR-2) and downregulation of fibrosis markers (α-SMA and vimentin). These results demonstrate that miR-218-5p and miR-363-3p mimics promoted CF angiogenesis and proliferation by promoting mesenchymal-endothelial transition in CFs.
Increasing numbers of targets regulated by miRNAs have been reported in MI, such as PPAR-γ, Smad7, bak1, klf13, and TXNIP [64][65][66]. In the present study, we found that p53 and JMY were directly regulated by miR-218-5p and miR-363-3p. In addition, miR-218-5p was found to activate p53 expression, and these results are consistent with previous research [67]. p53 and JMY have been demonstrated to participate in angiogenesis and proliferation in many diseases, especially in cancer [68][69][70]. However, the function of p53 and JMY in MI is rarely studied. In the present study, we found that suppression of p53 and overexpression of JMY could attenuate the CF angiogenesis and proliferation affected by miR-218-5p and miR-363-3p. In addition, evidence from other groups has shown a switch regulation between p53 and MEndT in CFs, which in the absence of p53 reduces the formation of fibroblast-derived endothelial cells and leads to cardiac injury, while activation of p53 promotes MEndT, resulting in cardiac function improvement [71]. Interestingly, in addition to the involvement of transcription cofactors in the p53 signaling pathway, JMY decreases the expression of adhesion molecules in the cadherin family and contributes to assembly of the actin cytoskeleton and to nucleation of new filaments [72]. Cadherins are endothelial-specific markers, and thus, a decrease in cadherin expression reflects a decrease in endothelial-mesenchymal transition (EMT) [18]. Taken together, these results demonstrate that upregulation of p53 by miR-218-5p and downregulation of JMY by miR-363-3p might relieve MF by inducing MEndT.
In addition, we identified the effect of exosomal miR-218-5p and miR-363-3p in MI by constructing an LCA ligationinduced MI rat model. The results indicated that arrhythmia, LVEDd, LVESd, LVIDs, HR, LVIDd, SV, LVEF, FS, CO, LVPWT, and LVAWT were partially restored by injection of EPC-Exos containing miR-218-5p and miR-363-3p mimic. Further analysis showed that EPC-Exos containing miR-218-5p and miR-363-3p mimic effectively suppressed infarction-induced myocardial damage by inhibiting inflammation and macrophage infiltration, downregulating collagen expression, and increasing capillary density, as shown by histochemical staining and analysis of collagen-1, collagen-3, and Timp-1-4 expression. These results demonstrate that exosomal miR-218-5p and miR-363-3p can reduce the cardiac dysfunction in MI rats and might provide new targets for MI therapy. However, future animal studies are required to guidance administration of miR-218-5p and miR-363-3p mimic to treat MI in the clinical context. Furthermore, the specific synthesis mechanism and function of exosomes are not very detailed and there is a lack of cost-effective exosome separation technology. In addition, future studies need to classify whether miRNA packaging into exosomes and exosomal uptake is a selective/stimulus-dependent process because there is no evidence to suggest functional differences between exosomal miRNAs and free ones and whether exosomal and free miRNAs are differentially regulated in response to stimulation. However, although the current biology of exosomes is still immature, more and more interest and capital investment will accelerate the progress of basic research and clinical transformation of exosomes.
Several limitations should be addressed in this study. Firstly, the expression of miRNAs in the EPC-Exos has been detected by qRT-PCR, while the localization of these miRNAs in the EPC-Exos may still require future experiments for validation. Secondly, the present study only examined two miRNAs (miR-218-5p and miR-363-3p) based on the microarray data, and future studies should carry out to further explore the functional roles of other potential miRNAs based on the microarray screening. Thirdly, the MI animal model was the nonreperfused ischemic injury model, and future studies may investigate the role of EPC-Exos in the reperfused ischemic injury model.