SU1498

Blocking exosomal miRNA-153-3p derived from bone marrow mesenchymal stem cells ameliorates hypoxia-induced myocardial and microvascular damage by targeting the ANGPT1-mediated VEGF/PI3k/Akt/eNOS pathway

Wenlong Ning1,2#, Shuhua Li4,5#, Weiguang Yang1,2, Bo Yang4,5, Chuanyou Xin1,2, Xin Ping1,2, Chuanqi Huang1,2, Yan Gu1,2, Longzhe Guo1,2,3*
1. Department of Emergency, the First Hospital of Qiqihar, Qiqihar 161005, Heilongjiang, China;
2. Department of Emergency, Affiliated Qiqihar Hospital, Southern Medical University, Qiqihar 161000, Heilongjiang, China;
3. Department of Anatomy, School of Basic Medical Sciences, Harbin Medical University, Harbin 150081, Heilongjiang, China;
4. Department of Traditional Chinese Medicine, the First Hospital of Qiqihar, Qiqihar 161005, Heilongjiang, China;
5. Department of Traditional Chinese Medicine, Affiliated Qiqihar Hospital, Southern Medical University, Qiqihar, Heilongjiang, China.

Abstract

It has been widely reported that exosomes derived from mesenchymal stem cells (MSCs) have a protective effect on myocardial infarction (MI). However, the specific molecules which play a damaging role in MSCs shuttled miRNAs are much less explored. MiRNA-153-3p (miR-153-3p) is a vital miRNA which has been proved to modulate cell proliferation, apoptosis, angiogenesis, peritoneal fibrosis and aortic calcification. Here, we aim to study the effect and mechanism of miR-153-3p in MSC-derived exosomes on hypoxia-induced myocardial and microvascular damage. The exosomes of MSCs were isolated and identified, and the MSCs-exosomes with low expression of miR-153-3p (exo-miR-153-3p-) were constructed to interfere with the endothelial cells and cardiomyocytes in the oxygen-glucose deprivation (OGD) model. The viability, apoptosis, angiogenesis of endothelial cells and cardiomyocytes were determined. Additionally, ANGPT1/VEGF/VEGFR2/PI3K/Akt/eNOS pathway was detected by ELISA and/or western blot. The results illustrated that exo-miR-153-3p- significantly reduced the apoptosis of endothelial cells and cardiomyocytes and promoted their viability. Meanwhile, exo-miR-153-3p- can promote the angiogenesis of endothelial cells. Mechanistically, miR-153-3p regulates the VEGF/VEGFR2/PI3K/Akt/eNOS pathways by targeting ANGPT1. Intervention with VEGFR2 inhibitor (SU1498, 1 μM) remarkably reversed the protective effect of exo-miR-153-3p- in vascular endothelial cells and cardiomyocytes treated by OGD. Collectively, MSCs-derived exosomes with low-expressed miR-153-3p notably promotes the activation of ANGPT1 and the VEGF/VEGFR2 /PI3K/Akt/eNOS pathways thereby preventing the damages endothelial cells and cardiomyocytes against hypoxia.

Keywords: myocardial damage; exosome; mesenchymal stem cells; miR-153-3p; ANGPT1.

Introduction

Myocardial infarction (MI) is pathologically defined as myocardial cell death due to long-term ischemia, which is a severe manifestation of coronary artery disease (CAD), with its risk factors including previous cardiovascular disease (CVD), diabetes and hypertension (1). In the past two decades, the rapid growth of domestic per-capita income and aging population in China have led to profound changes in population and epidemiology, and CVD has become a major non-communicable disease (2). Timely reconstruction of the occluded artery blood supply is the key to MI treatment. Usually, antithrombotic drugs, percutaneous coronary intervention and bypass surgery are used to treat patients; however, although these treatments can reduce the severity of CAD, they cannot restore infarction cardiac contractile force (3). Therefore, reducing myocardial cell death has good therapeutic prospects and would be very valuable for further improving patient prognosis.
Mesenchymal stem cells (MSCs), which are cells considered to have a multipotent differentiation potential, high transplantation and low immunogenicity, have a high therapeutic value in a variety of diseases, such as acute renal failure, bone and cartilage regeneration, neurological diseases and endothelial cell (EC) injury (4). Studies have reported that exosomes, a type of membrane vesicle ~100 nm in size, can release a variety of proteins and non-coding RNAs (ncRNAs) such as microRNAs (miRNAs) and lncRNAs through endocytosis or fusion with plasma membranes, thus regulating cell functions (5). In the last few decades, the effects of MSC-derived exosomal miRNAs on reducing cardiac cell death or stimulating cardiac cell regeneration have received increasing attention (6). The exosomes-shuttled miRNAs bind to the 3’-untranslated region of the target mRNA and negatively regulate it at the post-transcriptional level, thereby affecting the intracellular transmission of biological information (7).
In recent years, MSCs have been reported to improve the treatment of MI with its exosomes-shuttled miRNAs. For example, Xin et al found that, in a rat model of stroke, MSC-derived miR-133b considerably relieved neurological damage and promoted neuron regeneration in rats suffering from middle cerebral artery occlusion (MCAO) (8). Moreover, Xiao et al discovered that MSCs inhibited the expression of PDCD4 protein through miR-21, which inhibits the apoptosis of cardiomyocytes and protects cardiomyocytes against oxidative damage (9). miRNA-153-3p, a relatively new miRNA, has been rarely reported. Zhang et al reported that miRNA-153-3p regulated the autophagy and chemotherapy sensitivity of non-small cell lung cancer by targeting ATG5 (10). In addition, miR-153 was also found to be involved in regulating the functions of stem cells, such as maintained neural stem/progenitor cells (11), human MSCs (hMSCs) (12) and umbilical cord-derived MSCs (13). Furthermore, miR-153 has been shown to promote inflammation and oxidative stress-mediated cell damage (14-16). However, the role and mechanism of MSC exosomal miR-153-3p in hypoxia-induced cardiomyocytes remains virtually unknown.
Angiopoietin-1 (ANGPT1) plays an essential regulatory role between the endothelium and endothelium-matrix, promoting the maturation of immature vessels induced by vascular endothelial growth factor (VEGF), maintaining vascular structure, stabilizing ECs, inhibiting cell apoptosis and promoting the growth of ECs (17). ANGPT1 also has the effect of attracting perivascular cells and endothelial smooth muscle cells to accumulate and interact with one another, enhancing the connections between vascular ECs, thus helping to maintain the integrity of the vascular wall, promoting vascular remodeling (18). Therefore, ANGPT1 can be regarded as a supporting and stabilizing factor of ECs.
It was found in our previous study that the overexpression of miR-153-3p promoted EC damage and downregulated ANGPT1. In addition, even though MSCs exerted protective effects over ECs/cardiomyocytes, whether miR-153-3p in MSC-derived exosomes could mediate EC/cardiomyocyte damage is unknown. Herein, the level of miR-153-3p in MSC-derived exosomes was interfered with miR-153-3p inhibitor, and the effects of exo-miR-153-3p- on oxygen-glucose deprivation (OGD)-induced ECs and cardiomyocytes were explored. The present data suggested that MSC exosomal miRNA-153-3p regulated EC/cardiomyocyte damage by targeting the ANGPT1 pathway.

Materials and methods

Experimental animals. The animal experiments were approved by the ethics committee of the First Hospital of Qiqihar (Heilongjiang, China). The experimental procedures were in strict accordance with the guidelines for the management and use of laboratory animals formulated by the National Institutes of Health. Male C57BL/6 mice (age, 7-9 weeks; weight, 20-23 g) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China) and kept in sterile equipment. The mice were kept under a 12-h light/12-h dark cycle and standard conditions, with no limitation to water and food.

MSCs culture and identification. Mice were put to death after anesthesia, the bilateral legs were separated, the rest of the tissues excluded and the legs cut off at both ends. Next, the marrow cavity was repeatedly rinsed with a pre-cooled culture medium using a 1-ml syringe until the born turned pale. Next, the rinsing solution used was collected and underwent centrifugation at 300g for 5 min. After the suspension was removed, the cells were resuspended in high-glucose DMEM (Thermo Fisher Scientific, Inc.) and seeded in a culture dish. After 24 h, the supernatant was removed. Next, PBS was used to wash away the cells that did not adhere to the wall, and new culture medium was added. When the cells grew and covered 80-85% of the culture dish area, the passage was identified. After the 3rd passage generation, MSCs were fused to 80%, and the single-cell suspension was prepared following trypsinization and was divided into Eppendorf tubes. Samples to be tested were added with 100 μl PBS each and went through resuspension. Next, after the addition of 10 μL primary antibodies CD34, CD44, CD45, CD90 and CD105 (Cell Signaling Technology, Inc.) into the samples, they were fully mixed, incubated in the dark (30 min, room temperature) and washed twice with PBS (400 x g/5 min). Later, the supernatant was discarded, and 500 μl PBS was added to each test tube, thoroughly mixed and detected by flow cytometry.

Exosome isolation and transmission electron microscope (TEM) analysis. The exosomal separation procedure was carried out at 4˚C. Briefly, the supernatant of MSCs in different groups was filtered through a 0.2-m filter (10,000 x g/30 min) and then centrifuged at 100,000 x g for 3 h. The particles were re-suspended in 30-50 μl PBS and stored at -80˚C for other uses. The exosomes obtained as described above were negatively stained with uranyl acetate for mvar carbon coating in a 400-mesh copper electron microscope mesh (FCF400 Cu; Electron Microscopy Sciences) for morphological studies of TEM. The 20 μl sample was placed on the mesh and incubated at room temperature for 5 min. The excess solution was then removed from the mesh and dried with filter paper for 30 min. An equal amount of 10% uranyl acetate was added to the mesh for 5 min of negative staining. A Phillips 208 electron microscope (Philips Healthcare) and AMT digital imaging system (Advanced Micromicroscopy Technologies Corp.) under 70 KV voltage were used to check the preparation. The protein concentration of exosome preparation was determined by the BSA method.

Primary EC culture. The culture method of the primary EC was summarized as follows:

The 4-week-old mice were sacrificed after anesthesia, and the aorta was isolated and placed into a petri dish containing sterile D-Hanks solution (Thermo Fisher Scientific, Inc.) in a sterile state. After removing the fat and fibrous tissue and blood clot of the vascular outer membrane, the blood vessels were cut longitudinally, spread in the petri dish, cut into 1-mm3 artery grafts, and implanted into an incubation bottle containing 1% gelatin (preset overnight, 37˚C, planting density 1 piece/cm2). The cells were placed in the incubator (dry adherence) for 2 h, and then 0.5 ml culture medium containing 25% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) was added. After 24 h, the medium was changed and 2-3 ml medium was added. At ~72 h, when the ECs had fully grown and the fibroblasts were about to grow, the artery graft was removed, the fibroblasts were scraped, and the culture medium was changed once. The medium was then changed every 2 days, 1/2 to 1/3 each time. The cells were further cultured for 8-10 days until they fused into the monolayer.

Culture of cardiomyocytes H9c2. Rat cardiomyocyte H9c2 cells were purchased from the cell library of the Chinese Academy of Sciences. H9c2 was stored in DMEM medium (Thermo Fisher Scientific, Inc.) containing L-glutamine (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 100 mg/ml streptomycin (Sinopharm Chemical Reagent Co., Ltd.) and 10% FBS (Thermo Fisher Scientific, Inc.). The cells were incubated in a 5% CO2 incubator humidified at 37˚C. The OGD model was used to induce myocardial cell injury.

Cell transfection and treatment. The miR-153-3p inhibitors, miR-153-3p mimics and negative controls (NC-in or miR-NC) were synthesized by Guangzhou RiboBio Co., Ltd. The MSCs were inoculated into a 6-well plate (1×105 cells/well) 24 h before transfection. Next, the miR-153-3p inhibitors and NC-in were transfected into the primary MSCs, ECs or H9c2 cells using Lipofectamine 2000 Reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions. After a 24-h incubation, the culture medium was replaced with fresh culture medium and the cells were kept cultured for another 24 h. VEGFR2 inhibitor SU1498 (MedChemExpress) was used to block VEGFR2 at a concentration of 10 μM 30 min before the OGD model was established, according to a previous study.

OGD model establishment in vitro. MI often leads to ischemia/hypoxia-reperfusion injury. Hence, an OGD model was established to simulate the microenvironment of ECs and cardiomyocytes in vitro. Briefly, ECs and H9c2 cells in the logarithmic growth phase were collected and washed 3 times with glucose-free DMEM (Thermo Fisher Scientific, Inc.) balanced with 1% O2, 5% CO2 and 94% N2 in an incubator at 37˚C. The original medium was then replaced with glucose-free DMEM, and the cells were transferred to an incubator containing 1% O2, 5% CO2, and 94% N2 for 1.5 h at 37˚C. Later, the medium was replaced with the original medium, and the culture was returned to the standard incubator with a recovery time of 6 h. The control group was cultured for the same amount of time in a normal culture medium in an incubator with an atmosphere of 5% CO2 and viability was measured 24 h later.

CCK8 assay. Cell viability was assessed by CCK-8 assay. The ECs and H9c2 cells were inoculated into 96-well plates (1×103 cells/well). When the cells were adhered to the wall, they were treated with OGD and/or EXO-NC-in/EXO-miR-in. In accordance with the manufacturer’s instructions, 10 μl CCK8 was added to the treated cells (Dojindo Molecular Technologies, Inc.) reagent and underwent 1 h of incubation at 37˚C. The optical density value (450) was measured on a spectrophotometer (Bio-Rad Laboratories, Inc.).

Flow cytometry. Cell apoptosis was detected by flow cytometry using the Annexin V-FITC/PI staining kit (BD Biosciences). The treated cells were washed with cold PBS and resuspended in binding buffer (100 mmol/l NaCl, 25 mmol/l CaCl2,100 mmol/l HEPES; pH7.4). The cells were then stained with annexin V-FITC/PI in the dark (15 min, room temperature). Finally, flow cytometry was performed using a flow cytometer (BD FACS Calibur; Becton, Dickinson and Company) to evaluate the apoptosis rate. Each experiment was repeated three times.

Dual-luciferase reporter assay. The luciferase reporter vectors ANGPT1-wt and ANGPT1-mut were constructed by Shanghai GenePharma. 293T cells were inoculated in a 48-well plate and cultured to 70% confluence. ANGPT1-wt and ANGPT1-mut vectors were co-transfected with miRNA-153-3p mimics or negative control (miR-NC) using Lipofectamine 2000 Reagent (Thermo Fisher Scientific, Inc.). The luciferase viability was determined 48 h after transfection, according to the manufacturer’s instructions. All experiments were conducted in triplicate and repeated three times.

RT-PCR. Trizol (Tiangen Biotech Co., Ltd.) was used to extract the total RNA tissues and cells, according to the manufacturer’s instructions. After the purity and concentration of RNA were detected, RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.) was used for RT. The specific amplification primers of qPCR are shown in Table I, with U6 and GAPDH as endogenous control genes of miR-153-3p and ANGPT1, respectively. SYBR® Premix Ex Taq™ (Takara Bio, Inc.) was used to conduct the PCR experiments using Bio-Rad CFX96 (Bio-Rad Laboratories, Inc.), according to the manufacturer’s instructions. Each sample went through three repeated experiments, and the results were analyzed by the 2-ΔΔCT method. The primers used in this experiment are shown in Table I.

Western blotting. Total protein was extracted and protein concentration was detected using the BCA protein concentration kit (Beyotime Institute of Biotechnology). A total of 20 μg protein in each group was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 120 min at a voltage of 90V. Next, the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes under 200 mA constant current for 90 min. After blocking with 10% skimmed milk powder solution for 2 h, the PVDF membranes underwent overnight incubation with anti-CD9 (cat. no., ab92726, 1:1000), anti-CD81 (cat. no., ab79559, 1:1000), anti-TSG101 (cat. no., ab125011, 1:1000), anti-Caspase-3 (cat. no., ab197202, 1:1500), anti-Bax (cat. no., ab182733, 1:1000), anti-Bcl-2 (cat. no., ab32124, 1:2000), anti-VEGFA (cat. no., ab46154, 1:1000), anti-VEGFR2 (cat. no., ab11939, 1:1000), anti-PI3K (cat. no., ab154598, 1:1000), anti-p-PI3K (cat. no., ab182651, 1:1000), anti-e-NOS (cat. no., ab76198, 1:1000), anti-pe-NOS (cat. no., ab76199, 1:1000) and anti-β-Actin (cat. no., ab8226, 1:2000; Abcam, UK) primary antibodies at 4˚C. Next, the membranes were washed with TBST 3 times (15 min each) and incubated for 2 h with the secondary antibodies at room temperature. Next, the membranes were washed with TBST 3 times. Pictures were captured and the gray value of the strip was analyzed with Image Quant Las 4000 mini software (GE Healthcare). The target gene expression level was reflected by the ratio of the target gene band gray value to the β-actin band gray value.

Flow cytometry. Cells in the logarithmic growth phase were digested with 2.5 g/l trypsin, and then inoculated into 6-well culture plates with 1×106 cells per well. After 24 h, the original culture solution was aspirated and cells were treated as previously described. After centrifugation (1,500 rpm/min, 4˚C) for 5 min, the supernatant was discarded. The cells were re-suspended with PBS and counted. Next, ~1.5×105 cell suspension was centrifuged (1,500 rpm/min, 4˚C) and then resuspended. 1 x Binding buffer (400 μl, diluted in distilled water, 10 Binding Buffer) was added, and the mixture was gently suspended. Incubation was performed for 20 min in the dark at room temperature. Next, 5 μl PI was added and mixed gently, and flow cytometry was performed within 1 h. The excitation wavelength was Ex=488 nm and the emission wavelength Em=530 nm.

ELISA. The supernatant of each group was collected and used for indicator determination, and the ELISA kits of VEGF [(EK0541)/ANGPT1(EK1296)] (Wuhan Boster Biological Technology, Ltd.) were used to test the contents of VEGF and ANGPT1, respectively. The specific operation steps were carried out according to the manufacturer’s instructions. Briefly, samples or standards were added to the wells, followed by antibody mix. After incubation, the wells were washed to remove the unbound material. TMB Development Solution was added, and during incubation was catalyzed by HRP, generating blue coloration. This reaction was then stopped by adding STOP Solution completing any color change from blue to yellow. The signal was generated proportionally to the amount of bound analyte and the intensity was measured at 450 nm.

Determination of reactive oxygen species (ROS) activity. After the treatment of cells with drugs, the EC and cardiomyocytes were fixed with 10% formaldehyde (10 min, room temperature) and then washed with PBS 3 times (10 min each). Next, they were permeated with 0.5% Triton X-100 (5 min, room temperature), washed with PBS 3 times, sealed with 10% serum (1 h, room temperature) and then rewashed with PBS 3 times. Later, the cells were treated with Cellular ROS Assay Kit/Reactive Oxygen Species Assay Kit (cat. no., ab113851; Abcam), following the manufacturer’s instructions. Finally, the cells were detected by fluorescence spectroscopy with excitation/emission at 485/535 nm and pictures were captured.

Bioinformatics analysis. To investigate the underlying mechanism of miRNA-153-3p, the potential target genes of miR-153-3p were predicted through online databases, including miRmap. The targeted mRNAs of miR-153-3p and the common molecules of these databases were analyzed by Venn diagram.

Statistical analysis. Data are expressed as the mean ± standard deviation, statistical software Prism GraphPad 7.0 was used for statistical analysis, and the differences between groups were compared by one-way analysis of variance (ANOVA) or t-test. P<0.05 was considered to indicate a statistically significant difference. Results MSC-derived exosome identification. MSC medium supernatants were collected, and their stem cell characteristics were identified via flow cytometry. CD44, CD90 and CD105 are possible adhesion molecules that play a role in early hematopoiesis by mediating the attachment of stem cells to the bone marrow (BM) extracellular matrix or directly to stromal cells. The results showed that MSC-associated CD19, CD34 or CD45 antigens were negative, and the purified cells presented positive CD44, CD90 and CD105 phenotypes, which were consistent with the characteristics of MSCs (Fig. 1A). Next, the exosomes in the supernatant of MSCs were isolated by ultracentrifugation and observed by electron microscopy. It was found that exosomes were enriched and mainly distributed at 50-150 nm (Fig. 1B-C). Meanwhile, western blotting was performed to detect the expression of surface markers CD9, CD81 and TSG101 in MSCs and the isolated exosomes, and the results illustrated that CD9, CD81 and TSG101 were highly expressed in exosomes (Fig. 1D). Moreover, CD9, CD81 and TSG101 were upregulated in exosomes, when compared with non-exosome components of the media (Fig. 1E). The above results confirmed the successful isolation of the exosomes in MSCs. MSC exosomes with a low miR-153-3p expression attenuate OGD-mediated ECs injury. For the purpose of investigating the effect of miR-153-3p from MSC exosomes on ECs damaged by OGD, OGD-induced ECs were treated with exo-miR-153-3p-. RT-PCR results suggested that OGD treatment clearly enhanced the miR-153-3p level in ECs, as compared with the control group, while exo-miR-153-3p- considerably reduced miR-153-3p expression, as compared with the OGD-exo-NC-in group (Fig. 2A). Next, CCK8 assay and flow cytometry were used to test cell viability and apoptosis. The results suggested that OGD notably inhibited EC viability and increased apoptosis, while exo-miR-153-3p- promoted EC cell proliferation and inhibited apoptosis (Fig. 2B-C). In addition, western blotting showed that the expression of Bax and Caspase-3 were increased and Bcl-2 was decreased after OGD intervention, while exo-miR-153-3p- could reverse these effects (Fig. 2D). Moreover, the results of tubule formation assay revealed that tubule formation was decreased in OGD-induced ECs, but was increased exo-miR-153-3p- treatment (Fig. 2E). Next, the ROS level in ECs was detected usina Cellular ROS Assay Kit, and it was found that ROS levels were increased in OGD-induced ECs, but were decreased after exo-miR-153-3p- treatment (Fig. 2F). These results suggested that MSC-derived exosomes with a low miR-153-3p expression notably inhibited OGD-induced cell apoptosis and oxidative stress in ECs, as well as promoted angiogenesis. MSC exosomes with a low miR-153-3p expression promote ANGPT1 expression and VEGF/VEGFR2/PI3K/Akt/eNOS pathway activation in ECs. When further exploring the mechanism of miRNA-153-3p, it was found that exo-miR-153-3p- significantly improved the expression of ANGPT1 in the OGD model (Fig. 3A-B). Besides, ELISA and western blotting results showed that exo-miR-153-3p- considerably upregulated the expression of VEGF and VEGFR2 in OGD-treated ECs and increased the phosphorylation of PI3K, Akt and eNOS (Fig. 3C-D). The results indicated that exo-miR-153-3p- promoted the expression of ANGPT1 and activation of the VEGF/VEGFR2/PI3K/Akt/eNOS pathway in ECs. MSAs-Exo with a low miR-153-3p expression attenuates OGD-mediated cardiomyocyte injury. Next, OGD-induced cardiomyocytes were treated with MSC exosomes with a low miR-153-3p expression to investigate the protective effects of miR-153-3p inhibition on cardiomyocytes. First, RT-PCR results revealed that exo-miR-153-3p- markedly reduced the increased expression of miR-153-3p after OGD intervention in cardiomyocytes (Fig. 4A). The viability and apoptosis results suggested that OGD suppressed cardiomyocyte viability and increased cell apoptosis, while exo-miR-153-3p- promoted cell viability and inhibited apoptosis (Fig. 4B-D). Moreover, the ROS levels in cardiomyocytes were significantly increased after OGD intervention, but were decreased after treatment with exo-miR-153-3p- in cardiomyocytes (Fig. 4E). These results suggested that MSC exosomes rich in miR-153-3p inhibitor could inhibit OGD-induced myocyte apoptosis and oxidative stress. MSC exosomes with a lower expression of miR-153-3p promote ANGPT1 expression and VEGF/VEGFR2/PI3K/Akt/eNOS pathway activation in cardiomyocytes. To further explore the mechanism of miR-153-3p in cardiomyocytes, the level of ANGPT1 and VEGF, and the VEGFR2/PI3K/Akt/eNOS pathway were detected in cardiomyocytes by RT-PCR, ELISA and western blotting. The results indicated that OGD treatment obviously reduced ANGPT, VEGF and VEGFR2/PI3K/Akt/eNOS pathway activation in cardiomyocytes (Fig. 5 A-D). However, exo-miR-153-3p- intervention notably increased the expression of ANGPT1, VEGF and VEGFR2 in cardiomyocytes, and promoted the phosphorylation of PI3K, Akt and eNOS (Fig. 5 A-D). The results showed that MSC exosomes with a lower miR-153-3p expression promoted the ANGPT1 expression and activation of the VEGF/VEGFR2/PI3K/Akt/eNOS pathway in cardiomyocytes. VEGFR2 inhibition attenuates the protective effect of exo-miR-153-3p- on ECs and cardiomyocytes. To further confirm the effects of the VEGFR2 pathway in exo-miR-153-3p--mediated protective effects on ECs and cardiomyocytes, VEGFR2 inhibitors (SU1498; 10 μM) were used to intervene in ECs and cardiomyocytes. The viability and apoptosis of ECs and cardiomyocytes were detected. The results suggested that SU1498 considerably suppressed cell viability and increased apoptosis (Fig. 6A-B). Moreover, the ROS level in cells were significantly increased with SU1498 intervention (Fig. 6C). Western blot results showed that SU1498 considerably increased the expression of Caspase 3 and Bax, and decreased Bcl2 expression in ECs (Fig. 6D) and cardiomyocytes (Fig. 6E). Further detection of phosphorylation of PI3K, Akt and eNOS revealed that SU1498 notably reduced the activation of PI3K, Akt and eNOS in both ECs (Fig. 6F) and cardiomyocytes (Fig. 6G). Thereby, these results confirmed that the inhibition of VEGFR2 attenuated the protective effect of exo-miR-153-3p- on ECs and cardiomyocytes. miR-153-3p targets ANGPT1. In order to explore the targeting relationship between miR-153-3p and ANGPT1, the online databases miRmap, microT, miRanda and Targetscan were used to analyze the targeted mRNAs of miR-153-3p, and the target and common molecules of these databases were analyzed by Venn diagram. The results showed that 97 of them were common molecules in all four databases (Fig. 7A-B). The targeting relationship between ANGPT1 and miR-153-3p was observed in the four databases, and the specific binding site between them is shown in Fig. 7C. Dual luciferase activity experiments illustrated that miR-153-3p inhibited the luciferase activity of ANGPT1-wt, but had no significant effect on ANGPT1-mut (Fig. 7D). Besides, the over-expression of miR-153-3p in ECs and cardiomyocytes indicated that the expression of ANGPT1 was considerably reduced, as compared with the control group (Fig. 7E-F). Therefore, we hypothesized that the low expression of MSC exosomal miR-153-3p attenuates OGD-mediated vascular ECs and myocardial cell injury by promoting the ANGPT1/VEGF/VEGFR2/PI3K/Akt/eNOS pathway (Fig. 7G). Discussion In recent years, non-coding RNA and the value of stem cells in acute MI (AMI) have received increasing attention. The results of the present study showed that the enrichment of miR-153-3p in MSC-derived exosomes had a significant regulatory effect on ECs and cardiomyocytes. The low expression of MSC exosomal miR-153-3p markedly promoted the activation of the ANGPT1 and VEGF/VEGFR2/PI3K/Akt/eNOS pathways in ECs and cardiomyocytes, as well as protected them against OGD induced injury. Excessive inflammatory response and oxidative stress have been found to be closely associated with OGD-induced EC and cardiomyocyte damage. Inflammatory cytokines, such as tumor necrosis factor-α, monocyte chemoattractant protein-1, interleukin (IL)-6 and IL-1β, directly lead to death-related autophagy and apoptosis of cardiomyocytes and ECs (19-20). Though activation of hypoxia-inducible factor/VEGF signaling can promote angiogenesis (21), VEGF treatment alone is often not enough to protect against oxidative stress and promote post-ischemic angiogenesis, whereas the combined treatment of IGFBP4 and VEGF effectively facilitates angiogenesis through repressing ischemic and oxidative injury (22). On the other hand, either endogenously or exogenously generated ROS exerts a crucial role in regulating angiogenesis, apoptosis and proliferation of cells. For example, curcumin analog A2, as a novel antiangiogenic agent, induces endothelial cell death via elevating NADH/NADPH oxidase-derived ROS (23). The metabolic abnormalities lead to increased intracellular reactive oxygen species (ROS), which restrains angiogenesis and activate a number of proinflammatory pathways (24). However, there are evidences shown that reactive oxygen species (ROS) enhances angiogenesis and prevents tissue injury (25). Hence, those studies indicate that ROS has a dual role in modulating angiogenesis and apoptosis (26-27). Recent studies have suggested that MSCs have the ability to differentiate into various types of cells, including cardiomyocytes, vascular ECs and vascular smooth muscle cells (28). MSCs have been widely used in preclinical and clinical studies for the treatment of AMI and ischemic heart failure (IHF) (29). Initially, the beneficial effect of BMMSCs in the treatment of AMI and IHF was thought to be their ability to differentiate into cardiomyocyte types, but recent evidence has suggested that the differentiation of BMMSCs seems to be limited. BMMSCs mainly exert cardioprotective effects through the secretion of cytokines, exosomes and other forms (30-31). Ju et al found that, after the induction of AMI in mice, C-MSC-Exo was injected into the myocardium to the edge of the infarction, and the heart exhibited an improved cardiac function for 1 month. The capillary density and number of Ki67-positive cells were notably increased (32). Liu et al reported that BMMSC-derived exosomes with a high expression of macrophage migration inhibitor factor reduced mitochondrial fragmentation, ROS generation and apoptosis in cardiomyocytes, and also enhanced cardiac function and alleviated cardiac remodeling under hypoxia/serum deprivation (33). Thus, MSCs play a protective role in MI by secreting exosomes. Herein, it was also found that MSC-derived exosomes markedly relieved OGD-induced damage on ECs and cardiomyocytes by repressing apoptosis and oxidative stress, and promoting viability and angiogenesis. Except for a large number of proteins, MSC exosomes are also rich in non-coding RNA. In recent decades, reports on altered miRNAs in exosomes and phagocytosis by vascular ECs, cardiomyocytes, osteoblasts and other cells gradually increase (34). Previous studies have proved that MSCs have an anti-apoptosis, cardiac regeneration, anti-cardiac remodeling, anti-inflammatory, neovascularization and anti-vascular remodeling effect through secreting exosomes rich in miRNAs, which is considered as a new potential molecular mechanism for MSC transplantation (35-36). Pan et al discovered that the overexpression of miR-126 in MSC exosomes had a protective effect on ischemia-reperfusion-induced EC damage and vascular damage, and promoted the expression of VEGF, EGF, PDGF and bFGF (37). Wang et al reported that the miR-223 exosome expression could reduce the levels of Sema3A and STAT3, and make a significant contribution to myocardial protection after MSC-induced sepsis (38). In addition, Zhu et al reported that miR125b-5p derived from HYPO-Exo after myocardial infarction promoted ischemic heart repair by improving myocardial cell apoptosis (39). miR-153 has been found to exert a prominent effect on the modulation of the development of several types of cancer, including bladder (40), gastric (41) and ovarian cancer (42). In addition, in a nutritional stress-induced intervertebral disc degeneration (IDD) model, miR-153-3p inhibited autophagy and IDD by targeting ATG5 (43). In the permanent MCAO model of SD rats, the miR-153-3p level was downregulated in the infraction areas, but lncRNA HIF1A-AS2, HIF-1α, VEGFA and Notch 1 were increased. The forced expression of HIF1A-AS2 promoted the angiogenesis of human umbilical vein ECs by sponging miR-153-3p; this result indicated that miR-153-3p was a negative regulator of angiogenesis (6). Moreover, the knockdown of miR-153-3p inhibited mitochondrial fission and hypertrophy in isoprenaline-induced cultured primary cardiomyocytes (44). Of note, the MSCs containing miR-153-3p have also been found to play a role in modulating different diseases. For example, human umbilical cord MSCs (hUCMSCs) promoted the expression of miR-153-3p, thus attenuating MGO-induced peritoneal fibrosis in rats (45). In addition, miR-153-3p was upregulated in patients with systemic lupus erythematosus, and overexpressed miR-153-3p induced immune dysregulation of hUCMSCs by inhibiting PELI1 expression (46). It was found in our previous study that the high expression of miR-153-3p in human MSCs reduced the expression of ANGPT1 mRNA at the transcription level. Therefore, the therapeutic effects of MSC-derived exosomes with a low miR-153-3p expression were further explored. Of note, our data indicated that the downregulation of miR-153-3p in MSC-derived exosomes significantly enhanced the protective effects of MSC exosomes on OGD-induced ECs and cardiomyocytes. ANGPT1, also known as Ang1, has been identified as a vital modulator in regulating angiogenesis of ECs and cardiomyocyte damage (47-48). For example, Reis LA et al injected integrin-binding ANGPT1-derived peptide around or in the area of myocardial infarction in the rat model, which significantly improved cardiac function, scar thickness and scar area fraction, confirming the therapeutic value of ANGPT1 in AMI (49). Baumert et al reported that ANGPT1 is increased in the myocardium of patients with AMI and has a significant influence on prognosis (50). Cao Sheng et al proved that Ang1 could enhance left ventricular systolic function, increase myocardial perfusion, decrease fibrous tissue level and increase vascular density, but it had no significant effect on cardiac troponin I and n-terminal b-type natriuretic peptide levels (51). Parborell et al reported that ANGPT1 reduces cell apoptosis by activating the PI3K/Akt signaling pathway (52). A study has reported that ANGPT1 activated the Tie-2/Akt/eNOS signaling pathway in red blood cells, thus revealing a new biological mechanism that coincides with osmotic pressure reaction (53). Shujia Jiang et al confirmed that MSCs with Ang-1- and Akt-overexpression exhibited marked myogenic differentiation, improved neovascularization function and reduced deterioration of cardiac function (54). In addition, reports of miRNA regulation on ANGPT1 have been increasing in recent decades. Sabirzhanov et al reported that miR-711 directly targets the Ang-1 mRNA, decreases the expression of Ang-1, reduces the level of angiotensin-converting enzyme 1 after brain injury, aggravates the degeneration of neurons and damages the integrity of the blood-brain barrier (55). In the present study, ANGPT1 was found to be overexpressed, followed by miR-153-3p downregulation. Meanwhile, ANGPT1 upregulation was also consistent with the activation of the VEGF/VEGFR2/PI3K/Akt/eNOS pathway. However, inhibiting VEGFR2 markedly enhanced the apoptosis of ECs and cardiomyocytes, and inactivated the PI3K/Akt/eNOS pathway. It was then proven that miR-153-3p was a direct target of ANGPT1 and restrained ANGPT1 expression both in ECs and cardiomyocytes. Collectively, these results confirmed that the downregulation of miR-153-3p in MSC exosomes promotes the activation of the VEGF/VEGFR2/PI3K/Akt/eNOS pathway in ECs and cardiomyocytes by targeting ANGPT1. In conclusion, the results of this study suggested that MSC exosomes can be phagocytic by ECs and cardiomyocytes. Meanwhile, MSC exosomes with a low expression of miR-153-3p promote the activation of ANGPT1 and the VEGF/VEGFR2/PI3K/Akt/eNOS pathway, subsequently improving the function of the ECs and cardiomyocytes. Reference 1. Jafri, S. M. Antiplatelet and anticoagulant therapy in acute myocardial infarction. Henry Ford Hosp Med J. 39(3-4):195-9 (1991). 2. Zheng Yunting,Wu Yiqun,Wang Mengying et al. Impact of a comprehensive tobacco control policy package on acute myocardial infarction and stroke hospital admissions in Beijing, China: interrupted time series study.[J) .Tob Control, 2020, DOI：10.1136/tobaccocontrol-2020-055663. 3. Yeh, R. W., Kereiakes, D. J., Steg, P. G., Windecker, S., Rinaldi, M. J., Gershlick, A. H., Cutlip, D. E., Cohen, D. J., Tanguay, J. F., Jacobs, A., Wiviott, S. D., Massaro, J. M., Iancu, A. C., and Mauri, L. Benefits and Risks of Extended Duration Dual Antiplatelet Therapy After PCI in Patients With and Without Acute Myocardial Infarction. J Am Coll Cardiol. May 26;65(20):2211-21 (2015). 4. Tsiapalis Dimitrios,O’Driscoll Lorraine,Mesenchymal Stem Cell Derived Extracellular Vesicles for Tissue Engineering and Regenerative Medicine Applications.[J] .Cells, 2020, 9: 10.3390/cells9040991. 5. Hessvik Nina Pettersen,Llorente Alicia,Current knowledge on exosome biogenesis and release.[J] .Cell. Mol. Life Sci., 2018, 75: 193-208. 6. Zhu Ling-Ping,Tian Tian,Wang Jun-Yao et al. Hypoxia-elicited mesenchymal stem cell-derived exosomes facilitates cardiac repair through miR-125b-mediated prevention of cell death in myocardial infarction.[J] .Theranostics, 2018, 8: 6163-6177. 7. Fernandez-Valverde, S. L., Taft, R. J., and Mattick, J. S. MicroRNAs in beta-cell biology, insulin resistance, diabetes and its complications. Diabetes. Jul;60(7):1825-31 (2011). 8. Xin, H., Li, Y., Liu, Z., Wang, X., Shang, X., Cui, Y., Zhang, Z. G., and Chopp, M. MiR-133b promotes neural plasticity and functional recovery after treatment of stroke with multipotent mesenchymal stromal cells in rats via transfer of exosome-enriched extracellular particles. Stem Cells. Dec;31(12):2737-46 (2013). 9. Xiao, J., Pan, Y., Li, X. H., Yang, X. Y., Feng, Y. L., Tan, H. H., Jiang, L., Feng, J., and Yu, X. Y. Cardiac progenitor cell-derived exosomes prevent cardiomyocytes apoptosis through exosomal miR-21 by targeting PDCD4. Cell Death Dis. Jun 23;7(6):e2277 (2016). 10. Zhang W,Dong Y-Z,Du X et al. MiRNA-153-3p promotes gefitinib-sensitivity in non-small cell lung cancer by inhibiting ATG5 expression and autophagy.[J] .Eur Rev Med Pharmacol Sci, 2019, 23: 2444-2452. 11. Tsuyama J, Bunt J, Richards LJ, et al. MicroRNA-153 Regulates the Acquisition of Gliogenic Competence by Neural Stem Cells. Stem Cell Reports. 2015;5(3):365-377. 12. Cao Y, LV Q, LV C. MicroRNA-153 suppresses the osteogenic differentiation of human mesenchymal stem cells by targeting bone morphogenetic protein receptor type II. Int J Mol Med. 2015;36(3):760-766. 13. Li D, Li X, Duan M, et al. MiR-153-3p induces immune dysregulation by inhibiting PELI1 expression in umbilical cord-derived mesenchymal stem cells in patients with systemic lupus erythematosus. Autoimmunity. 2020;53(4):201-209. 14. Sun Y, Zhou S, Shi Y, et al. Inhibition of miR-153, an IL-1β-responsive miRNA, prevents beta cell failure and inflammation-associated diabetes. Metabolism. 2020;154335. 15. Hou W, Zhu X, Liu J, Ma J. Inhibition of miR-153 ameliorates ischemia/reperfusion-induced cardiomyocytes apoptosis by regulating Nrf2/HO-1 signaling in rats [published correction appears in Biomed Eng Online. 2020 Jun 9;19(1):43. Map, Jiaguo [corrected to Ma, Jiaguo]]. Biomed Eng Online. 2020;19(1):15. 16. Zhu J, Wang S, Qi W, Xu X, Liang Y. Overexpression of miR-153 promotes oxidative stress in MPP+-induced PD model by negatively regulating the Nrf2/HO-1 signaling pathway. Int J Clin Exp Pathol. 2018;11(8):4179-4187. 17. Kim Kyun Hoo,Nakaoka Yoshikazu,Augustin Hellmut G et al. Myocardial Angiopoietin-1 Controls Atrial Chamber Morphogenesis by Spatiotemporal Degradation of Cardiac Jelly.[J] .Cell Rep, 2018, 23: 2455-2466. 18. Gilfillan Margaret,Das Pragnya,Shah Dilip et al. Inhibition of microRNA-451 is associated with increased expression of Macrophage Migration Inhibitory Factor and mitgation of the cardio-pulmonary phenotype in a murine model of Bronchopulmonary Dysplasia.[J] .Respir. Res., 2020, 21: 92. 19. Wang X, Guo Z, Ding Z, Mehta JL. Inflammation, Autophagy, and Apoptosis After Myocardial Infarction. J Am Heart Assoc. 2018;7(9):e008024. 20. Vila V, Martínez-Sales V, Almenar L, Lázaro IS, Villa P, Reganon E. Inflammation, endothelial dysfunction and angiogenesis markers in chronic heart failure patients [published correction appears in Int J Cardiol. 2011 Feb 3;146(3):484]. Int J Cardiol. 2008;130(2):276-277. 21. Kim YW, Byzova TV. Oxidative stress in angiogenesis and vascular disease. Blood. 2014;123(5):625-631. 22. Wo D, Chen J, Li Q, et al. IGFBP-4 enhances VEGF-induced angiogenesis in a mouse model of myocardial infarction [published online ahead of print, 2020 Jun 28]. J Cell Mol Med. 2020;24(16):9466-9471. 23. Liu B, Cui LS, Zhou B, Zhang LL, Liu ZH, Zhang L. Monocarbonyl curcumin analog A2 potently inhibits angiogenesis by inducing ROS-dependent endothelial cell death. Acta Pharmacol Sin. 2019 Nov;40(11):1412-1423. 24. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res. 2010 Oct 29;107(9):1058-70. 25. Wang Z, Yang J, Qi J, Jin Y, Tong L. Activation of NADPH/ROS pathway contributes to angiogenesis through JNK signaling in brain endothelial cells. Microvasc Res. 2020 Sep;131:104012. 26. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007;39(1):44-84. 27. Wong W. New connections: The duality of ROS in angiogenesis. Sci Signal. 2017;10(479):eaan6438. 28. Wang Chen,Jing Qing,Non-coding RNAs as biomarkers for acute myocardial infarction.[J] .Acta Pharmacol. Sin., 2018, 39: 1110-1119. 29. Chen Fang,Liang Peng,Ye Fang et al. Mesenchymal stem cell therapy for patients with ischemic heart failure -past, present, and future.[J] .Curr Stem Cell Res Ther, 2020, 10.2174/1574888X15666200309144906. 30. Suzuki Etsu,Fujita Daishi,Takahashi Masao et al. Therapeutic Effects of Mesenchymal Stem Cell-Derived Exosomes in Cardiovascular Disease.[J] .Adv. Exp. Med. Biol., 2017, 998: 179-185. 31. Shafei Ayman El-Sayed,Ali Mahmoud Ahmed,Ghanem Hazem G et al. Mesenchymal stem cell therapy: A promising cell-based therapy for treatment of myocardial infarction.[J] .J Gene Med, 2017, 19: 10.1002/jgm.2995. 32. Ju Chengwei,Shen Yan,Ma Gengshan et al. Transplantation of Cardiac Mesenchymal Stem Cell-Derived Exosomes Promotes Repair in Ischemic Myocardium.[J] .J Cardiovasc Transl Res, 2018, 11: 420-428. 33. Liu Xiaolin,Li Xin,Zhu Wenwu et al. Exosomes from mesenchymal stem cells overexpressing MIF enhance myocardial repair.[J] .J. Cell. Physiol., 2020, 10.1002/jcp.29456. 34. Teng Xiaomei,Chen Lei,Chen Weiqian et al. Mesenchymal Stem Cell-Derived Exosomes Improve the Microenvironment of Infarcted Myocardium Contributing to Angiogenesis and Anti-Inflammation.[J] .Cell. Physiol. Biochem., 2015, 37: 2415-24. 35. Huang Lina,Ma Wenya,Ma Yidi et al. Exosomes in mesenchymal stem cells, a new therapeutic strategy for cardiovascular diseases?[J] .Int. J. Biol. Sci., 2015, 11: 238-45. 36. Li Bo,Luan Song,Chen Jing et al. The MSC-Derived Exosomal lncRNA H19 Promotes Wound Healing in Diabetic Foot Ulcers by Upregulating PTEN via MicroRNA-152-3p.[J] .Mol Ther Nucleic Acids, 2020, 19: 814-826. 37. Pan Qunwen,Wang Yan,Lan Qing et al. Exosomes Derived from Mesenchymal Stem Cells Ameliorate Hypoxia/Reoxygenation-Injured ECs via Transferring MicroRNA-126.[J] .Stem Cells Int, 2019, 2019: 2831756. 38. Wang Xiaohong,Gu Haitao,Qin Dongze et al. Exosomal miR-223 Contributes to Mesenchymal Stem Cell-Elicited Cardioprotection in Polymicrobial Sepsis.[J] .Sci Rep, 2015, 5: 13721. 39. Zhang W, Mao S, Shi D, et al. MicroRNA-153 Decreases Tryptophan Catabolism and Inhibits Angiogenesis in Bladder Cancer by Targeting Indoleamine 2,3-Dioxygenase 1. Front Oncol. 2019;9:619. 40. Ouyang Y, Yuan W, Qiu S. MicroRNA-153 functions as a tumor suppressor in gastric cancer via targeting Kruppel-like factor 5. Exp Ther Med. 2018;16(2):473‐ 482. 41. Li C, Zhang Y, Zhao W, Cui S, Song Y. miR-153-3p regulates progression of ovarian carcinoma in vitro and in vivo by targeting MCL1 gene. J Cell Biochem. 2019;120(11):19147‐19158. 42. Wang XB, Wang H, Long HQ, Li DY, Zheng X. LINC00641 regulates autophagy and intervertebral disc degeneration by acting as a competitive endogenous RNA of miR-153-3p under nutrition deprivation stress. J Cell Physiol. 2019;234(5):7115‐ 7127. 43. Li L, Wang M, Mei Z, et al. lncRNAs HIF1A-AS2 facilitates the up-regulation of HIF-1α by sponging to miR-153-3p, whereby promoting angiogenesis in HUVECs in hypoxia. Biomed Pharmacother. 2017;96:165‐172. 44. Wang T, Zhai M, Xu S, et al. NFATc3-dependent expression of miR-153-3p promotes mitochondrial fragmentation in cardiac hypertrophy by impairing mitofusin-1 expression. Theranostics. 2020;10(2):553‐566. 45. Li D, Lu Z, Li X, et al. Human umbilical cord mesenchymal stem cells facilitate the up-regulation of miR-153-3p, whereby attenuating MGO-induced peritoneal fibrosis in rats. J Cell Mol Med. 2018;22(7):3452‐3463. 46. Li D, Li X, Duan M, et al. MiR-153-3p induces immune dysregulation by inhibiting PELI1 expression in umbilical cord-derived mesenchymal stem cells in patients with systemic lupus erythematosus [published online ahead of print, 2020 Apr 23]. Autoimmunity. 2020;1‐9. 47. Nowicki M, Wierzbowska A, Małachowski R, et al. VEGF, ANGPT1, ANGPT2, and MMP-9 expression in the autologous hematopoietic stem cell transplantation and its impact on the time to engraftment. Ann Hematol. 2017;96(12):2103‐2112. 48. Dallabrida SM, Ismail N, Oberle JR, Himes BE, Rupnick MA. Angiopoietin-1 promotes cardiac and skeletal myocyte survival through integrins. Circ Res. 2005;96(4):e8‐e24. 49. Reis Lewis A,Chiu Loraine L Y,Wu Jun et al. Hydrogels with integrin-binding SU1498 angiopoietin-1-derived peptide, QHREDGS, for treatment of acute myocardial infarction.[J] .Circ Heart Fail, 2015, 8: 333-41.
50. Baumert Bartłomiej, Przybycień Krzysztof, Paczkowska Edyta et al. Novel Evidence of the Increase in Angiogenic Factor Plasma Levels after Lineage-Negative Stem/Progenitor Cell Intracoronary Infusion in Patients with Acute Myocardial Infarction.[J] .Int J Mol Sci, 2019, 20: 10.3390/ijms20133330.
51. Cao Sheng,Zhou Qing,Chen Jin-Ling et al. Comparison of Intracoronary and Intravenous Ultrasound-targeted Microbubble Destruction-mediated Ang1 Gene Transfection on Left Ventricular Remodeling in Canines With Acute Myocardial Infarction.[J] .J. Cardiovasc. Pharmacol., 2017, 70: 25-33.
52. Parborell Fernanda,Abramovich Dalhia,Irusta Griselda et al. Angiopoietin 1 reduces rat follicular atresia mediated by apoptosis through the PI3K/Akt pathway.[J] .Mol. Cell. Endocrinol., 2011, 343: 79-87.
53. Chu You-Hsiang,Lu Chih-Cherng,Lin Tso-Chou et al. The Osmopressor-Induced Angiopoietin-1 Secretion in Plasma and Subsequent Activation of the Tie-2/Akt/eNOS Signaling Pathway in Red Blood Cell.[J] .Am. J. Hypertens., 2017, 30: 295-303.
54. Shujia Jiang,Haider Husnain Khawaja,Idris Niagara Muhammad et al. Stable therapeutic effects of mesenchymal stem cell-based multiple gene delivery for cardiac repair.[J] .Cardiovasc. Res., 2008, 77: 525-33.
55. Sabirzhanov Boris,Faden Alan I,Aubrecht Taryn et al. MicroRNA-711-Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death.[J] .J. Neurotrauma, 2018, 35: 2462-2481.