Peripheral Serum Exosomes Isolated from Patients with Acute Myocardial Infarction Promote Endothelial Cell Angiogenesis via the miR-126-3p/TSC1/mTORC1/HIF-1α Pathway

Purpose Angiogenesis is required for improving myocardial function and is a key factor in long-term prognosis after an acute myocardial infarction (AMI). Although exosomes are known to play a crucial role in angiogenesis, the role of peripheral exosomes in angiogenic signal transduction in patients with AMI remains unclear. Here, we explored the effect of exosomes extracted from the peripheral serum of AMI patients on angiogenesis and elucidated the downstream pathways. Patients and Methods Serum exosomes were obtained from patients with AMI (AMI-Exo) and healthy individuals (Con-Exo). The exosomes were cocultured with human umbilical vein endothelial cells (HUVECs) in vitro, with aortic rings ex vivo, and were used to treat mouse hind-limb ischemia and mouse AMI model in vivo. Results AMI-Exo raised HUVEC proliferation, tube formation, and migration, and enhanced microvessel sprouting from aortic rings compared to Con-Exo, both in vitro and ex vivo. Quantitative reverse transcription-polymerase chain reaction revealed that the abundance of miR-126-3p, a crucial regulator of angiogenesis, was increased in AMI-Exo. The inhibition of miR-126-3p decreased the benefits of AMI-Exo treatment, and miR-126-3p upregulation enhanced the benefits of Con-Exo treatment in HUVECs, aortic rings, the mouse hind-limb ischemia model, and the mouse AMI model. Knockdown and overexpression analyses revealed that miR-126-3p regulated angiogenesis in HUVECs by directly targeting tuberous sclerosis complex 1 (TSC1). Moreover, we found that miR-126-3p could inhibit TSC1 expression, which further activated mTORC1 signaling and increased HIF-1α and VEGFA expression, ultimately promoting angiogenesis. Conclusion Collectively, our results provide a novel understanding of the function of exosomes in angiogenesis post AMI. We demonstrated that exosomes from the peripheral serum of AMI patients promote angiogenesis via the miR-126-3p/TSC1/mTORC1/HIF-1α signaling pathway.


Introduction
Cardiovascular diseases (CVDs) pose immense health and economic burdens worldwide, and account for 44% of all disease-related deaths in China. 1 Acute myocardial infarction (AMI) is the leading cause of death from CVDs. Although thrombolytic anticoagulant therapy and percutaneous coronary intervention can largely restore occluded coronary perfusion after AMI, they cannot repair the damage caused to microvascular architecture in ischemic myocardium by

Experimental Animals
Wild-type male 8-9-week-old C57BL/6 mice and 6-week-old Sprague-Dawley rats were purchased from the Medical Laboratory Animal Supply Base (Heilongjiang, China). The animals were reared in accordance with the principles of animal care approved by the National Society for Medical Research and the Guideline for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources/NIH). The experiments on animals were allowed by the Research Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (SYDW2020-030).

Quantitative RT-PCR Analysis
Exosomal RNA was derived from exosomes using a miRcute miRNA Isolation Kit (Tiangen Biotech, Beijing, China) and then reverse transcribed into cDNA using an miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech), based on standard protocols. Total cellular RNA was extracted by TRIzol reagent (Invitrogen) and mRNA was reverse transcribed using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Risch-Rotkreuz, Switzerland). For exosomal miRNA, the relative levels were normalized compared to those of synthetic Caenorhabditis elegans (cel)-miR-39 (5 fmol/mL; Applied Biosystems, California, USA). Meanwhile, for cellular miRNA, relative levels were normalized with U6 RNA. Cellular mRNA levels were normalized to GAPDH mRNA standards. Quantitative analysis was conducted using the 2 −ΔΔCt method. The primers are shown in Table S1.

HUVEC Proliferation, Tube Formation, and Migration
HUVEC proliferation was evaluated using Cell Counting kit-8 (Dojindo, CK04-11, Japan), Cell-Light EdU (5-ethynyl-2′deoxyuridine) kit (Ribobio, C10310-3, Guangzhou, China), and manual cell counting assay. Briefly, 100 µL of cell suspension including approximately 3000 HUVECs was placed in a 96-well plate and treated with 200 μg/mL AMI-Exo, Con-Exo, and PBS, respectively. After 24 h, CCK-8 reagent (10 μL) was added and the cells were cultivated for 3 h. Then, spectrophotometric microplate reader was used for measuring the optical density (OD) at 450 nm. In the EdU assay, the labelling reagent was added to a 96-well plate and the HUVECs were allowed to proliferate for 2 h. The cells were imaged using a fluorescence microscope (Leica, Germany).
HUVEC migration was analyzed using Transwell inserts (Corning, 3422, USA) and scratch wound assays. Cells were resuspended in serum-free DMEM, dispensed in the Transwell inserts (approximately 2×10 5 cells/well); the cells then migrated to the underside containing DMEM with 10% FBS, through the 8.0 µm pore polycarbonate membrane. The cells were then treated with PBS, Con-Exo, AMI-Exo, and cultured for 8 h at 37°C with 5% CO 2 . Cells within each insert were removed using cotton swabs. The migrated cells were treated with crystal violet for 10 min and quantitated in three random microscopic fields.
Scratch wound assays were carried out by resuspending HUVECs seeded in 6-well plates and starving them for 12h. A scratch wound was created using a yellow pipette tip and then, PBS, Con-Exo or AMI-Exo were added, respectively. After 24 h, the wound area was observed and analyzed by ImageJ software. The ratio of non-migrated area divided by the baseline area was used to calculate the migration effects.
In in-vitro tube formation assay, 50 μL Matrigel Matrix (BD, 356234) was added to a 96-well plate. Then, about 2×10 4 cells/well were seeded on the Matrigel and co-cultured with PBS, Con-Exo, or AMI-Exo for 8 h. The total tube length and number of nodes were quantitatively analyzed by ImageJ software. Six-week old Sprague-Dawley rats were euthanized and their thoracic aorta dissected. After the removal of the surrounding tissue and fat, the aorta was cut into~1 mm sections. One ring was gently placed in each well of a 96-well plate with 70 μL mixed Matrigel in serum-free medium 1:1, and incubated at 37°C. Ring sprouting was visualized on day 4 using a light microscope with a phase-contrast setting. Sprout outgrowths were counted manually. The number of sprouts that emerged from the main ring as well as the individual branches arising from it as separate vessels were counted. 10

Luciferase Assay
Overlap analysis revealed that TSC1 may be a potential therapeutic target. Wild-type and mutated TSC1 mRNA 3ʹ UTR fragments were synthesized and inserted into the p-MIR-REPORT plasmid (GenePharma, Shanghai, China). The 293T cells were co-transfected with 60 nM of mimic miR-126-3p or inhibitor and 10 ng of Renilla luciferase reporter. After 24 h, Renilla luciferase activity was evaluated by Dual-Luciferase Reporter Assay System (Promega, USA), based on the manufacturer's protocol.

Statistical Analysis
Continuous variables were presented as the mean ± standard deviation or as the median and interquartile range. Student's t-test or Mann-Whitney U-test, as appropriate. Categorical variables were expressed as numbers and percentages, and analyzed using the chi-square test. The SPSS statistical software (version 25.0; SPSS Inc., IBM Corp., Armonk, NY, USA) was employed. Two-sided P values of < 0.05 were considered as statistically significant. Histograms were drawn using GraphPad Prism 8.0 (GraphPad Software, California, USA). Further information on the methods is described in the Supplementary Information.

Serum Exosome Characterization
Twenty AMI patients and 10 healthy individuals were included in this study. Their clinical baseline characteristics are shown in Table 1. Serum exosomes from the healthy individuals and AMI patients were derived by ultracentrifugation, as reported previously, 11 and their morphology was analyzed using TEM. The exosomes displayed a round, cup-shaped morphology ( Figure 1A). Specific exosomal markers (Alix, CD63, and TSG101) were expressed in both exosome types ( Figure 1B), and NanoSight measurement showed that they both displayed particle size distributions of 30-150 nm ( Figure 1C). Meanwhile, there was no statistical difference in concentration between Con-Exo and AMI-Exo ( Figure 1D). Taken together, these results confirm that exosomes were the main components of the purified microvesicles.

Serum-Derived Exosomes Were Internalized Well by Endothelial Cells
To explore whether the exosomes can be internalized by endothelial cells, we incubated PKH67-stained exosomes with HUVECs for 24 h; > 90% of HUVECs were found to be positive for PKH67 fluorescence, with no difference between the internalization of AMI-Exo and Con-Exo ( Figure 1E). Confocal imaging confirmed that the exosomes were effectively internalized by the HUVECs ( Figure 1F). Thus, the isolated exosomes could be internalized by endothelial cells.

AMI-Exos Promote Angiogenesis in vitro and ex vivo
To explore the functions of the exosomes acting on endothelial cells, HUVECs were co-cultured with exosomes isolated from both (control and AMI) groups. AMI-Exo promoted HUVEC proliferation to a greater extent than Con-Exo after treatment, as revealed by EdU and CCK8 assays (Figure 2A-C) and confirmed by manual cell counting ( Figure 2D). Vascular formation effect was evaluated using Matrigel assays, which showed that AMI-Exo augment the total tube length and number of nodes compared to Con-Exo, indicating that they possess higher pro-angiogenic abilities ( Figure 2E-G). To assess the migration of cells after exosome treatment, we also performed Transwell and scratch https://doi.org/10.2147/IJN.S338937

DovePress
International Journal of Nanomedicine 2022:17 wound assays. AMI-Exo exerted greater effects than Con-Exo on both the migration area in the wound assay ( Figure 2H and I) and the migrated cell number in the Transwell assay ( Figure 2J and K). To further investigate the pro-angiogenic properties of the AMI exosomes, we performed aortic ring assays, which revealed that conditioned media contained

1581
AMI-Exo markedly enhanced microvessels sprouting from aortic rings ( Figure 2L and M). Overall, results of our in vitro and ex vivo assays showed that both Con-Exo and AMI-Exo could promote angiogenesis compared with PBS group. However, AMI-Exo exerted greater effects than Con-Exo in angiogenesis-related procedures, including proliferation, tube formation, migration, and microvessel sprouting, suggesting that exosomes from the peripheral serum of patients with AMI may contain certain angiogenic signals than those from healthy individuals.

miR-126-3p Expression is Up-Regulated in AMI-Exo
Recent studies have demonstrated that exosomes exert crucial effects in different cell types via miRNA transport. To investigate the mechanisms underlying the angiogenic roles of the exosomes, we chose several angiogenesis-related miRNAs (miR-143, miR-17-5p, miR-21-5p, miR-939-5p, and miR-126-3p), 5,12-15 and validated their differential expression in the serum exosomes of AMI patients and control individuals using qRT-PCR. miR-126-3p expression differed significantly between the two groups, indicating that circulating AMI-Exos may load miR-126-3p ( Figure 3A-E). Next, HUVECs were incubated with exosomes and RNA polymerase II inhibitor. The results showed that there was no significant difference in the miR-126-3p expression between the two groups ( Figure 3F), suggesting that AMI-Exo did not induce endogenous miR-126-3p transcription in HUVECs. The effect induced by miR-126-3p, thus was attributed to exosome transport.

miR-126-3p from AMI-Exo Promotes Angiogenesis in vitro and ex vivo
To verify that miR-126-3p is essential for the exosomal regulation of angiogenesis in vitro and ex vivo, we co-cultured HUVECs and aortic rings with PBS, Con-Exo + mimic NC, Con-Exo + mimic miR-126-3p, AMI-Exo + inhibitor NC, and AMI-Exo + inhibitor miR-126-3p. Con-Exo + mimic miR-126-3p and AMI-Exo + inhibitor NC promoted endothelial cell proliferation better than Con-Exo + mimic NC and AMI-Exo + inhibitor, respectively, as indicated by CCK8 and Edu analyses ( Figure 4A-D). In addition, Matrigel assays to assess vascular formation ability revealed that Con-Exo transfected with the miR-126-3p increased the number of nodes and total tube length, whereas AMI-Exo transfected with the inhibitor miR-126-3p decreased AMI-Exo induced vascular formation, suggesting that exosomal miR-126-3p exerts pro-angiogenic effects post AMI ( Figure 4E-G). We also performed Transwell and scratch wound assays to evaluate the migration of cells considering the four types of exosomes. Notably, exosomal miR-126-3p transfection promoted cell migration in wound assay ( Figure 4H and I) and increased the number of migrated cells in Transwell assay ( Figure 4J and K). While, exosomal inhibitor miR-126-3p transfection abolished the effects of AMI-Exo promoted HUVEC migration ( Figure 4H-K). Furthermore, conditioned media contained Con-Exo + mimic miR-126-3p or AMI-Exo + inhibitor NC markedly enhanced microvessel sprouting from aortic rings ( Figure 4L and M). Together, these findings indicate that miR-126-3p in AMI-Exos plays a key role in angiogenesis.

miR-126-3p from AMI-Exo Promotes Angiogenesis in Mouse Hind Limb Ischemia
To further investigate the angiogenic effects of miR-126-3p from AMI-Exo and Con-Exo in vivo, we generated a mouse model of unilateral hind limb ischemia that was injected intramuscularly with PBS or exosomes on day 1 after surgery. As per a previous study, 5 exosomes were diluted in PBS. Then, we injected 200μL different concentration AMI-Exo, as 5×10 2 μg/mL, 1×10 3 μg/ mL, 2×10 3 μg/mL, or PBS on day 1 after surgery. We found that the exosomal concentration of approximately 1×10 3 μg/mL in 200 μL PBS was the optimal amount ( Figure S1). Thus, this amount was chosen for the following experiments. qRT-PCR assays revealed a high miR-126-3p expression in the muscles treated with Con-Exo + mimic miR-126-3p and AMI-Exo + inhibitor NC ( Figure 5A). No significant differences in blood flow recovery were observed between the four groups 3 and 7 days posttreatment; however, the Con-Exo + mimic miR-126-3p and AMI-Exo + inhibitor NC groups showed significantly higher blood perfusion 14 days post-injection than the Con-Exo + mimic NC and AMI-Exo + inhibitor miR-126-3p groups, respectively ( Figure 5B and C). The gastrocnemius muscles were harvested, embedded, sectioned, and subjected to H&E staining ( Figure 5D) and immunofluorescence ( Figure 5E). Angiogenesis after ischemia was detected using the vascular marker CD31 and α-SMA. The groups treated with Con-Exo + mimic miR-126-3p and AMI-Exo + inhibitor NC showed a significantly higher capillary density than those treated with Con-Exo + mimic NC and AMI-Exo + inhibitor miR-126-3p ( Figure 5F), respectively, indicating https://doi.org/10.2147/IJN.S338937

miR-126-3p from AMI-Exo Promotes Angiogenesis in Mouse AMI Model
To further investigate the angiogenic effects of exosomal miR-126-3p in mouse AMI model, we intramyocardially injected PBS or exosomes into the left ventricular wall (border zone) immediately after the left anterior descending ligation. The cardiac tissue was collected 14 days after the myocardial infarction. The qRT-PCR assays revealed that the miR-126-3p-enriched exosomes could effectively deliver miR-126-3p to the myocardium ( Figure 6A). The immunohistochemical assay was performed to assess angiogenesis after ischemia by detecting the number of vessels. Con-Exo + mimic miR-126-3p group showed a significantly higher capillary density than Con-Exo + mimic NC group. The AMI-Exo + inhibitor miR-126-3p group showed a significantly lower capillary density than AMI-Exo + inhibitor NC group ( Figure 6B-D). Collectively, these analyses of the mouse AMI model showed that miR-126-3p in peripheral serumderived exosomes promotes post-ischemic endothelial cell angiogenesis.

miR-126-3p Promotes Endothelial Cell Angiogenesis by Targeting TSC1 and Activates the mTORC1/HIF-1α Signaling Pathway
Mutations in TSC1 or TSC2 have been reported to lead to benign malformations or tumors, such as lymphangioleiomyomatosis and angiomyolipoma, in several different organs. 16 Therefore, TSC1 may play important roles in angiogenesis. The starBase, miRWalk, miRcode, and miRDB databases predicted that TSC1 is a target gene of miR-126-3p ( Figure 7A). The qRT-PCR analyses validated that miR-126-3p overexpression greatly suppresses TSC1 expression in HUVECs, with miR-126-3p inhibition exerting the opposite effects ( Figure 7B). To investigate the binding between miR-126-3p and the 3ʹ UTR of TSC1, we performed luciferase assays in which the TSC1 wild-type 3ʹ UTR or mutant 3ʹ UTRs were cloned into a reporter plasmid and their response to miR-126-3p was assessed in HUVECs ( Figure 7C). The mimic miR-126-3p markedly decreased luciferase activity, whereas the inhibitor miR-126-3p increased luciferase activity. Moreover, the mutant 3ʹ UTRs of TSC1 did not alter the response of luciferase activity to the mimic miR-126-3p or inhibitor ( Figure 7D). Therefore, these results confirm that TSC1 is a direct target of miR-126-3p. Further Western blot analyses suggested that miR-126-3p overexpression reduces TSC1 expression, with miR-126-3p inhibition boosting TSC1 expression. TSC1 is known to target the mTORC1/HIF-1α pathway. 17 Hence, our next step was to confirm the effects of changes in miR-126-3p expression on the TSC1/mTORC1/HIF-1α pathway. HUVECs treated with mimic of miR-126-3p had improved levels of phosphorylated S6 (pS6) and S6, which are markers of mTORC1 activation, compared to those treated with the control. Conversely, pS6 and S6 levels decreased when the cells were treated with the inhibitor of miR-126-3p. mTORC1 signaling was highly activated in HUVECs along with increase in HIF-1α and VEGFA levels ( Figure 7E and F), consistent with previous reports that mTORC1 signaling regulates HIF-1α transcription. 18 Subsequently, we constructed a TSC1 overexpression plasmid and simultaneously transfected with miR-126-3p. We found that the miR-126-3p induced mTORC1 activation and increased the expression of HIF-1α and VEGFA, which were abolished by TSC1 overexpression (Figure 7G and H).
To further investigate whether miR-126-3p could modulate angiogenesis by targeting TSC-1, we utilized validation experiment, wherein, miR-126-3p and TSC1 were overexpressed simultaneously to observe the phenotype of angiogenesis. The overexpression of TSC1 abolished miR-126-3p-promoted endothelial cell proliferation effect, as indicated by CCK8 and Edu analyses ( Figure 8A-D). In scratch wound assays, the overexpression of TSC1 could reduce the effect of Figure 5 Effect of miR-126-3p in AMI-Exo and Con-Exo on mouse ischemic injury after hind limb ischemia. (A) miR-126-3p expression in ischemic muscles was measured using qRT-PCR after treatment for 24 h (n = 3). (B) Blood flow perfusion was measured using laser Doppler imaging 1, 3, 7, and 14 days after the exosomes were treated with PBS, mimic miR-126-3p, mimic NC, inhibitor miR-126-3p, or inhibitor NC. The left paw was the ischemic and the right paw was normal control (n = 5). (C) The blood perfusion recovery was calculated as the perfusion ratio of the mean velocity in the left rear paw divided by that in the right rear paw. ***Means mimic miR-126-3p vs mimic NC, ### Means inhibitor miR-126-3p vs inhibitor NC (D) The gastrocnemius muscle sections were stained by hematoxylin and eosin. (E) α-SMA and CD31 were identified based on immunofluorescence (red: α-SMA, green: CD31, blue: DAPI). Arrows indicate microvessels. Scale bar, 100 μm. (F) Capillary density in ischemic muscles was measured by the number of microvessels per cm 2 . **p < 0.01, ***p < 0.001, ### p < 0.001. miR-126-3p-promoted cell migration area in wound assay ( Figure 8E and F). Furthermore, in aortic ring assay, the overexpression of TSC1 could blocked miR-126-3p induced microvessel sprouting ( Figure 8G and H). Collectively, these findings indicate that miR-126-3p promotes endothelial angiogenesis by targeting TSC1.

Discussion
Exosomes carry many functional messages which play key roles in cell-to-cell interactions during myocardial ischemia. MiRNAs are small noncoding RNAs with 22-23 nucleotides that could post-transcriptionally restrain gene expression by targeting the 3ʹ UTR of mRNAs. Notably, exosomal miRNAs have emerged as key mediators in diverse biological and pathological processes, including the promotion of angiogenesis and improvement of cardiac function. 18,19 Indeed, recent studies have verified that exosomes containing miRNAs can be delivered into other cells to post-transcriptionally regulate gene expression. 20 Exosomes from the coronary artery serum of patients with myocardial ischemia or those with myocardial infarction have been shown to promote angiogenesis through the miR-939-iNOS-NO pathway 5 and through the IGF-IR/NO signaling pathway. 21 However, exosomes secreted by cells may undergo diverse changes and exert different effects under different pathophysiological conditions, 22 and the effects of exosomes derived from the peripheral serum of patients with early stage AMI on angiogenesis remain unclear.
Zhang and Zhang 23 demonstrated that mouse cardiomyocytes could release specific exosomes into the bloodstream under ischemic conditions. Therefore, in this study we obtained exosomes from human peripheral serum, which were subsequently internalized by endothelial cells. We found that AMI-Exo enhanced HUVEC proliferation, tube formation, migration, and enhanced microvessel sprouting from aortic rings. It was recently discovered that exosomes contain numerous miRNAs, including miR-143 and miR-126, that participate in various phases of angiogenesis. 5,15,21 In particular, miR-126 is expressed at high levels in vascular endothelial cells and is known as a master regulator for vascular integrity and physiological angiogenesis. 24,25 Xue et al 26 reported that the expression levels of plasma miR-17-5p, miR-126, and miR-145-3p were significantly augmented in patients with AMI within 4 h of the onset of chest pain. Moreover, Ling et al 27 reported that serum exosomal miR-126 and miR-21 levels were higher in unstable angina (UA) and AMI patients (within 2 h of AMI) than healthy controls. Moreover, circulating exosomal miR-126 expression levels correlated positively with the severity of coronary artery stenosis defined by Gensini score. Therefore, in this current study, we validated the expression of some angiogenesis-related miRNAs, including miR-143, miR-17-5p, miR-21-5p, miR-939-5p, and miR-126-3p between two groups using qRT-PCR. Among them, the miR-126-3p expression levels were significantly upregulated in AMI-Exo. Moreover, gain-offunction analysis in HUVECs revealed that miR-126-3p significantly promoted cell proliferation, tube formation, migration, and microvessel sprouting. miR-126-3p also improved angiogenesis to a greater extent in AMI-Exo-treated cells than Con-Exo-treated cells in mouse limb ischemia and AMI models. Jakob et al 28 previously revealed that exosomal miR-126 from the endothelial cells of patients with chronic heart failure could affect angiogenesis by regulating SPRED1/VEFG signaling. In neonates, TSC1 loss has been shown to increase retinal angiogenesis and lead to endothelial proliferative impairment and generation of vascular tumors in mice by activating mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and increasing HIF-1α and VEGFA expression. 17 Although the post-transcriptional inhibition of TSC1 by miR-126-3p has been reported in granulosa cells, 29 the source and delivery route of miR-126-3p during the early stages of AMI remain unknown. Since bioinformatics analysis and a previous study 29 have hinted that TSC1 is a promising target gene of miR-126-3p, we used luciferase reporter assays to demonstrate that miR-126-3p directly targets the 3ʹ UTR of TSC1 and represses TSC1 expression.
TSC1 is a subtype of the TSC gene that can form a protein complex with TSC2. 30 The mutations in TSC1 and TSC2 have been shown to give rise to the lymphangioleiomyomatosis. 31 The complex formed by the TSC1 and TSC2 gene products is a pivotal negative regulator of mTORC1 and cell growth. 32 mTOR is a serine/threonine kinase which regulates a number of cellular processes, 33 and any abnormality in mTORC1 signaling can result in many diseases including vascular malformations and even tumors. 34,35 Studies have shown the key role of VEGF in tumor development regulated by HIF-1α, Figure 8 miR-126-3p-targeted TSC1 promotes angiogenesis in vitro. The mimic NC, mimic miR-126-3p, mimic miR-126-3p + oe. NC, and mimic miR-126-3p + oe. TSC1 were transfected into HUVECs. (A) HUVEC proliferation was measured by manual cell counting, CCK8 absorbance (B), and EdU staining (C and D). Scale bar, 50 μm. Cell migration was assessed by scratch wound assays (E) and analyzed as the ratio of non-migrated area divided by the baseline wound area (F). Scale bar, 100 μm. (G) Microvessels were quantified using aortic sprouting assays (H). Scale bar, 200 μm. **p < 0.01, ***p < 0.001.
International Journal of Nanomedicine 2022:17 https://doi.org/10.2147/IJN.S338937 DovePress which is present downstream of mTORC1, 17 and that HIF-1α is an essential mediator of VEGF-induced angiogenesis. 36,37 Consistently, we observed that miR-126-3p targeted TSC1, which activated the mTORC1 signaling pathway, increased pS6 and S6 expression, and increased HIF-1α and VEGFA expression. Interestingly, HIF-1α upregulation has been shown to affect the regulated miR-126 expression upstream that can promote angiogenesis and subsequently improve heart function in myocardial infarction. 38 Therefore, there may be a previously undescribed mechanism underlying the promotion of endothelial angiogenesis that involves a positive feedback loop of miR-126-3p and HIF-1α, and this should be verified in future studies.
Previously, Nie et al 39 reported that circulating miR-126-3p levels were significantly higher in the plasma from sufficient coronary collateral circulation in patients with severely narrowed coronary arteries. Unlike the targeting function of exosomal miRNA, circulating miRNA acts as a rover in the circulatory system that can react with any cell type that is in contact with the blood stream. Therefore, the findings of this study suggest that exosome-mediated miR-126-3p delivery to endothelial cells could better explain the improved coronary collateral circulation in patients with higher miR-126-3p levels.
However, this study also has some limitations. First, we focused on the expression of only few miRNAs in exosomes, which might have resulted in the neglect of other functional miRNAs present in patients with AMI. Second, because of the complicated contents of exosomes, the miR-126-3p/TSC1/mTORC1/HIF-1α pathway may be just one of multiple pathways that regulate angiogenesis.

Conclusion
In summary, our results revealed that miR-126-3p in exosomes derived from the peripheral serum of AMI patients can promote angiogenesis in endothelial cells by targeting TSC1 and activating the mTORC1/ HIF-1α axis. Together, the findings of this study provide a novel understanding of the function of exosomal miRNAs during the early stages of AMI onset.

Ethics Approval and Informed Consent
The research protocol was approved by the Second Affiliated Hospital of Harbin Medical University Ethics Committee (KY2021-147, SYDW2020-030).
We obtained written informed consent from all subjects.