Ethics statement
This study was performed following the approval of the Ethical Committee of Qingdao Hiser Medical Center. All the experiments were implemented on the guide for the care and use of laboratory animals.
Cell isolation and culture
To isolate EPCs, we used anti-CD133 coupled magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) to screen the cells expressing early EPC surface marker CD133 from the cord blood of C57BL/6 mice. Then, EPCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). The cell morphology was observed using an inverted microscope.
By referring to the modified method of Kobayashi et al., mouse aortic endothelial cells (MAECs) were isolated from the aorta of C57BL/6 mice. Briefly, the mice were dissected from the midline of the abdomen and the chest was opened. The aorta of mice was isolated and washed with phosphate-buffered saline (PBS). The aorta was cut longitudinally to remove the fat or connective tissues, followed by washing with serum-free DMEM (12100-46, GIBCO, Grand Island, NY, USA). Next, the aorta was incubated with collagenase II solution (171010115, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 45 min. Thereafter, the aorta was isolated from the adventitia and rinsed with DMEM (F8067, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) containing 20% FBS to collect MAECs. Next, the cells were centrifuged and cultured in DMEM containing 20% FBS at 37 °C for 2 h. Afterward, cells were rinsed with PBS and cultured in the fresh medium. After 1 week, the cells were passaged upon reaching 90% confluence. MAECs at passage 3 were used for subsequent experiments.
Immunofluorescence
EPCs were fixed with 40 g/L polyformaldehyde for 15 min, rinsed with PBS three times, blocked with 10% goat serum, and incubated with the primary antibodies CD34 (ab110643, Abcam Inc., Cambridge, MA, USA), CD133 (ab222782, Abcam), vWF (ab6994, Abcam), and VEGF-2 (ab2349, Abcam) at 4 °C overnight. Then, the cells were added with TRITC-labeled fluorescent secondary antibody goat anti-rabbit IgG H&L (ab6718, 1:1000, Abcam) and FITC-labeled fluorescent secondary antibody goat anti-mouse IgG H&L (ab6785, 1:1000, Abcam) for 45 min. Following two PBS washings, the cells were cultured with Hoechst3342 for 15 min, washed, and finally photographed using a fluorescent microscope.
Cell transfection and lentivirus infection
EPCs were infected with miR-199a-3p lentivirus silencing vector (anti-miR-199a-3p) and its negative control (anti-miR NC). MAECs were infected with specificity protein 1 (SP1) lentivirus overexpression vector (oe-SP1) and its negative control oe-NC, and transfected with NC mimic, miR-199a-3p mimic, NC inhibitor, or miR-199a-3p inhibitor.
For lentivirus infection, GFP-labeled miR-199a-3p lentiviral silencing vector and SP1 lentiviral overexpression vector (both purchased from Genechem, Shanghai, Shanghai) were transfected into 293T cells (CRL-1573, American Type Culture Collection, Manassas, VA, USA). From 24 h to 72 h after transfection, the supernatant containing lentivirus particles was collected every 12 h and filtered through a 0.45 μm cellulose acetate filter. The final virus titer was 2 × 1010 TU/mL. One day before lentivirus infection, EPCs and MAECs were seeded into the six-well plates (1 × 105 cells/mL). When the cell confluence reached 50–70%, the culture medium was sucked out. Each well was added with 2 × 104 TU complete culture medium containing 50-fold dilutions of the virus and 10 μg/mL Polybrene (H8761, Solarbio, Beijing, China) and cultured at 37 °C with 5% CO2. After 48–72 h of transfection, the cells stably infected with lentivirus were screened in the medium containing 0.5 μg/mL puromycin (A1113803, Invitrogen Inc., Carlsbad, CA, USA). The medium was refreshed every 3 days and the cell line with stable expression was obtained.
mimic NC (50 nM), miR-199a-3p mimic (50 nM), inhibitor NC (150 nM), and miR-199a-3p inhibitor (150 nM) were purchased from Ribobio (Guangzhou, China). ECs were seeded into the 24-well plates (5 × 105 cells/well) and transfected using Lipofectamine 3000 (Invitrogen). The final volume of each well was 500 μL.
Isolation and purification of EPC-EVs
The culture medium/serum was ultracentrifuged at 100,000 × g and 4 °C overnight to remove the EVs from the serum. EPCs were cultured in endothelial basal medium (EBM-2; Longza, Allendale, NJ, USA) supplemented with EBM-2 SingleQuots, 10% EV-free FBS (System Biosciences, Palo Alto, CA, USA), and 1% penicillin and streptomycin (GIBCO, Gaithersburg, MD, USA) for 48 h. The culture medium was collected and centrifuged at 500 × g and 4 °C for 15 min to eliminate cell debris, centrifuged at 2000 × g and 4 °C for 15 min to remove cell debris or apoptotic bodies, following centrifugation at 10,000 × g and 4 °C for 20 min to remove large vesicles. The cells were filtered with a 0.22 μM filter, centrifuged at 110,000 × g and 4 °C for 70 min, resuspended with PBS, and then ultracentrifuged under the same conditions, and finally resuspended with 100 μL sterile PBS.
Identification of EVs
For Nanosight nanoparticle tracking analysis (NTA), 20 μg EVs were dissolved in 1 mL PBS for 1 min to retain the uniform distribution of EVs. Then, EV particle size distribution was directly measured using a Nanosight nanoparticle tracking analyzer (Malvern Panalytical, UK).
For transmission electron microscopy (TEM), 20 μL ultracentrifuged EVs were loaded to carbon-coated copper electron microscope grids for 2 min, followed by negatively 5-min of staining with phosphotungstic acid solution (12501-23-4, Sigma-Aldrich). The grid was then washed with PBS three times to eliminate excess dye solution and kept semi-dry with filter paper. The images were observed using a TEM (H7650, Hitachi, Tokyo, Japan) at 80 kV.
The surface markers of EVs were identified using western blot. EV suspension was concentrated, and then, total protein concentration was examined using a bicinchoninic acid (BCA) assay kit (23227, Thermo Fisher Scientific). SDS-PAGE gel was prepared and protein denaturation and electrophoresis were performed. Afterward, the expressions of EV specific markers CD9 (ab92726, Abcam), CD81 (ab92726, Abcam), Alix (ab76608, Abcam), and calnexin (ab22595, Abcam) were detected.
Package of miR-199a-3p in EVs
RNase A treatment was used to determine whether miR-199a-3p was surface bound to or packaged in EVs. EVs were resuspended in PBS and then incubated with 2 μg/μL RNase A at 37 °C for 20 min. EV membrane integrity was destroyed using the detergent. After 20 min of 0.1% Triton X-100 treatment, the above RNase A treatment was carried out. After RNase A incubation, the lysis buffer was added to inhibit the reaction, and RNA was isolated.
Uptake of EVs
Purified EPC-EVs were labeled using a PKH67 green fluorescent kit (Sigma-Aldrich). EVs were resuspended in 1 mL Diluent C, and 4 × 10−6 M dye solution was prepared by adding 4 μL PKH67 ethanol dye solution to 1 mL Diluent C. Next, 1 mL EV suspension was stained with dye solution for 5 min and cultured with 2 mL 1% EV-free FBS for 1 min to terminate dyeing. EVs were enriched in the sucrose (1.13–1.19 g/mL) by centrifuging at 100,000 × g for 2 h [36]. PKH67-labeled EVs were treated with ECs for 12 h-incubation at 37 °C. The cells were fixed with 4% paraformaldehyde and washed with PBS, and the nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). As for the detection of the uptake of EPC-EVs carrying Cy3-miR-199a-3p by ECs, EPCs were transfected with Cy3-miR-199a-3p (GenePharma, Shanghai, China). After 6 h, the culture medium was changed to 10% EV-free serum medium for 48-h incubation. The supernatant was collected, centrifuged, resuspended with PBS, and added to ECs. Similarly, the cells were fixed with 4% paraformaldehyde, washed with PBS, and the cytoskeleton was labeled with Phalloidin-iFluor 488 Reagent (1:1000, ab176753, Abcam) at room temperature for 30 min. The nucleus was stained with DAPI. Finally, the uptake of EVs by ECs was observed under the confocal microscope (LSM710, Zeiss, Germany).
Oxidized low-density lipoprotein treatment
Oxidized low-density lipoprotein (ox-LDL) can induce cell death. MAECs were cultured in DMEM containing 20% FBS at 37c with 5% CO2. The cells were assigned into control group, ox-LDL group, EVs anti-NC group, EVs miR-199a-3p group, oe-NC group, oe-SP1 group, and oe-SP1 + EVs group. Briefly, the MAECs in the ox-LDL group were treated with 100 μg/mL ox-LDL, or co-treated with 100 μg/mL ox-LDL and 4 μL EVs (1 × 109 particles/mL) for 24 h.
MTT assay
Cell viability was assessed using MTT assay. Briefly, cells were seeded to the 96-well plates (2 × 103 cells/well) containing 100 μL complete medium. After co-incubation, each well was added with 20 μL MTT solution (5 mg/mL) at 37a for 4 h. Then, each well was added with 150 μL dimethyl sulphoxide and cultured at 37n for 20 min. The optical density (OD) value was read at a wavelength of 490 nm, and each group had three duplicated wells.
Detection of lactate dehydrogenase
The supernatant of cell culture was collected, and the activity of LDH was detected using the LDH assay kit (ab102526, Abcam). Briefly, 25 μL cell supernatant was mixed with 25 μL substrate and cultured at 37 °C for 15 min. Then, the sample was added with 2,4-dinitrophenylhydrazine at 37 °C for 15 min and cultured with 250 μL 0.4 M NaOH for 5 min. The absorbance was measured on the EvolutionTM spectrophotometer (840-210600, Thermo Fisher Scientific).
Trypan blue staining
Cells in the logarithmic growth phase with good growth conditions were obtained and centrifuged for 10 min at 1200 × g to remove the supernatant. Cells were added with an appropriate amount of culture medium and resuspended to obtain single cell suspension cultured in serum-free conditions. Cell concentration was calculated and adjusted to 4.0 × 106 cells/bottle. Cells were treated with EVs for 1 h at 37 °C. Subsequently, 900 μL cell fluid was collected after thorough mixture and added with 100 μL 0.4% trypan blue and mixed thoroughly, followed by observation under the microscope after 1-min staining. The blood cell counting plate was covered with the cover slip. One drop of stained cell suspension was dripped onto the top and bottom edges of the counting plate with a dropper to fill the gap between the blood cell counting plate and cover slip. Dead cells were observed and counted under the microscope and cell death rate was calculated. The experiment was repeated three times independently to obtain average value. Cell death rate (%) = dead cell number/total cell number × 100%.
Detection of glutathione and malondialdehyde
GSH (A006-2) and malondialdehyde (MDA) (A003-1) levels in the thoracic aorta tissue and MAECs were measured according to the manufacturer’s instructions of commercial kits. All the kits are purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, Jiangsu, China).
Iron determination
Total iron in cell lysate and tissue supernatant was quantified using an iron detection kit (ab83366, Abcam). In short, the particles of isolated tissues and cultured MAECs were homogenized with saline and PBS and centrifuged at 16,000 × g for 10 min to remove the insoluble matters. Then, 5 µL iron reducing agent was added to 50 µL sample to get the total iron content (Fe 3+and Fe 2+). Next, 100 µL iron probe solution was added to the sample and incubated at 25 °C in the dark for 60 min. The absorbance at 593 nm was measured using a spectrophotometer. Each experiment was repeated three times.
Detection of reactive oxygen species
The lipophilic fluorescent dye C11-BODIPY581/591 (D3861, Journal Pre-proof Gibco) was used to determine the lipid ROS level in MAECs. Briefly, after treatment and incubation, the cells were collected and washed with PBS. The lipid ROS was labeled with 5 μmol/L C11-BODIPY581/591 at 37 °C for 30 min. Following PBS washing three times, the cells were resuspended in 500 mL PBS. The fluorescence activity was analyzed using a flow cytometer (NovoCyte, Aceabio, San Diego, CA, USA). With unstained cells as the negative control and 50 μg/mL oxidant rosup-cultured (S0033S, Beyotime, Shanghai, China) cells as the positive control, ROS was detected by fluorescence.
Quantitative real-time polymerase chain reaction
The total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using Prime Script RT kit (Takara, Dalian, China). miRNA cDNA was synthesized from total RNA of cells and tissues using miRcute Plus miRNA First Strand cDNA synthesis kit (TianGen Biotech Co., Ltd. Beijing, China). The synthesized exogenous reference cel-miR-39 (1 pmol per sample; TianGen) was added to 350 µL culture medium or 100 µg EVs in advance. Real-time PCR of mRNA was performed using the SYBR Premix Ex Taq kit (Takara) and ABI StepOne real-time PCR system (Applied Biosystems, Inc., Carlsbad, CA, USA), with GAPDH as the internal reference. Real-time PCR of miRNA was conducted using miRcute Plus miRNA qPCR kit (TianGen), with U6 as the internal reference. In addition, the miRNA level in the medium and EVs were standardized according to the exogenous internal reference cel-miR-39. The relative transcription level of target genes was calculated using the 2-△△CT method. △△Ct = △Ct experimental group-△Ct control group, in which △Ct = Ct target gene-Ct internal reference gene. Ct is the number of amplification cycles when the real-time fluorescence intensity of the reaction reaches the set threshold. At this time, the amplification is logarithmic. Each experiment was repeated three times. The primer sequences are presented in Table 1.
Western blot
The protein was lysed in lysis solution supplemented with phosphatase inhibitor, protease inhibitor, and phenylmethylsulfonyl fluoride, followed by the measurement of protein concentration using a BCA kit (Thermo Fisher Scientific). Then, 10–20 µg protein was sampled on 8–12% 30% acrylamide-Bis gel and then transferred onto 0.22 µm PVDF membranes. Subsequently, the membranes were treated with 5% skim milk for 1 h and incubated with antibodies GAPDH (ab8245, Abcam), SP1 (ab227383, Abcam), GPX4 (ab125066, Abcam), and SLC7A11 (ab175186, Abcam) overnight. All antibodies were diluted according to the instructions. Next day, the membranes were cultured with the secondary antibody peroxidase-conjugated goat anti-rabbit IgG (H&L) (#111035003, Jackson ImmunoResearch, USA) for 1 h and developed with luminescent liquid (Thermo Fisher Scientific). Image J was used for analysis. The relative protein content was expressed as the gray value of the corresponding protein band/the gray value of GAPDH protein band. The experiment was repeated three times.
Dual-luciferase reporter assay
The synthetic vector Vector-SP1 3′UTR gene fragment was introduced into pmiRGLO dual-luciferase miRNA target expression vector (E1330, Promega, Madison, WI, USA). The mutation site of binding site was designed on the wild-type SP1. The target segment was inserted into pmiRGLO vector by T4 DNA ligase after restriction endonuclease. The above operations were completed by Genechem (Shanghai, China). The luciferase reporter plasmids were co-transfected with miR-199a-3p mimic into HEK293T cells. The cells were lysed after 48 h. The dual-luciferase reporter assay system kit (E1910, Promega) and Luminometer TD-20/20 instrument (E5311, Promega) were utilized to measure luciferase activity. The experiment was repeated three times.
RNA immunoprecipitation
RIP was performed using the Magna RIP kit (Millipore). In short, MAECs were lysed in radio-immunoprecipitation assay (RIPA) lysis buffer and centrifuged at 12,000 × g and 4 °C for 10 min to obtain the supernatant. One part of the cell extract was taken out as input, and the other part was incubated with antibody for co-precipitation. The specific steps were as follows: 50% μL magnetic beads in each co-precipitation reaction system were washed and suspended in 100 μL RIP wash buffer, and 5 μg antibody was added according to the experimental grouping for binding. The magnetic bead-antibody complex was washed and resuspended in 900 μL RIP wash buffer, supplemented with 100 μL cell extracts, and cultured at 4 °C overnight. The sample was placed on the magnetic stand for the collection of magnetic bead-protein complex. The sample and input were detached by proteinase K and then RNA was extracted for subsequent PCR detection. The antibody AGO2 (1:50, ab32381, Abcam) was mixed at room temperature for 30 min, and IgG (1:100, ab109489, Abcam) was used as NC.
Animal experiment
Male C57BL/6 mice and ApoE-/- mice aged 6–8 weeks were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and raised in a specific pathogen-free grade animal room at 20–22 °C with 40–60% humility and maintained in a 12 h light/dark cycle, with free food and water. The mice used in the experiment were kept in the animal room for at least 1 week.
Thirty ApoE-/- mice were fed an atherosclerotic diet (#D12108c, Research Diets Inc., New Brunswick, NJ, USA) containing high cholesterol (1.25%) to induce AS. After 4 weeks of feeding, 100 μg/kg EVs (EVs and EVs anti-miR 199a-3p) was injected into each mouse via tail vein every day for 3 days. After injection, the mice were raised in the animal experimental center. The remaining mice were injected with the same dose of PBS. The wild-type C57BL/6 mice served as normal controls (N = 10). After 2 weeks, the mice were anesthetized by pentobarbital sodium and then killed. The heart blood was collected by the cardiac puncture method. After PBS perfusion, the thoracic aorta was collected and fixed in 4% paraformaldehyde for 24 h, then placed in 30% sucrose-PBS buffer, embedded and frozen at the optimal cutting temperature.
Oil red O staining
Briefly, 10 μm fresh frozen sections were rinsed with 78% methanol for 1 min, and then 60 mL ORO working solution (Muto Pure Chemicals Co., Ltd., Bunkyo-ku, Japan) was added into distilled water to prepare a double dilution. After mixing and standing for 10 min, the sections were incubated with the diluent for 50 min. After that, the sections were incubated in 78% methanol, Meyer heme, and 0.05% lithium carbonate each for 1 min (the sections were washed with tap water after each step). The sections were observed and photographed after drying. The following formula was used to calculate the lesion area (%): ORO-positive staining area/total lesion area × 100%.
Enzyme-linked immunosorbent assay
Briefly, 3 mL heart blood of the mice was centrifuged for 15 min to collect the upper serum. The serum samples were kept in the dry sterile EP tube at −80 °C. The frozen serum samples were taken out and naturally dissolved at room temperature. The levels of TNF-α and IL-6 in mouse serum were detected using the ELISA kits. The absorbance (A) of each well at 450 nm was measured using the microplate reader (Synergy 2, BioTek). The regression equation of the standard curve was calculated with the standard concentration as abscissa and A value as ordinate. The A value of sample was substituted into the equation to calculate the concentration of target protein in the sample.
Statistical analysis
Statistical analyses were introduced using SPSS 21.0 (IBM Corp. Armonk, NY, USA) and GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). Measurement data are depicted as mean ± standard deviation. The unpaired t test was employed for the comparisons between two groups. One-way analysis of variance (ANOVA) was employed for the comparisons among multiple groups, followed by Tukey’s multiple comparisons test. A value of p < 0.05 was regarded statistically significant.
