Experimental materials
Rat bone marrow endothelial progenitor cells separation kits were bought from TBD company (China). CD34, and CD133 were purchased from Abcam (UK). Bax and Bcl2 were purchased from Proteintech (China). VCAM-1 was bought from Abcam (UK). ELISA kits for ET-1, TNF-α, IL-6 were purchased from ZCibio (China). Transferase dUTP nick end labeling (TUNEL) staining kit was purchased from Beyotime (China). Cleaved-caspase3 was purchased from Affinity (China). Rat bone marrow-derived endothelial progenitor cells complete medium was purchased from Procell (China). Endothelial cell growth medium-2 (EGM-2) was purchased from Lonza (Switzerland). LPS was purchased from Sigma (US). Mouse Monoclonal CD34 Antibody and Rabbit Polyclonal CD133 Antibody, used to flow cytometry, were bought from Univ (China). Cell culture medium was purchased from Pricella (China). Human Dil-labeled acetylated low-density lipoprotein (Dil-LDL) was purchased from MKbio (China). FITC labeled ulex europaeus agglutinin 1 (FITC-UEA-1) was purchased from Yiyuan Biotechnology (China). Ultrafiltration centrifugal tubes were purchased from Millipore (Germany). Ultracentrifuge tubes were purchased from Beckman (US).
Methods
Animal
All animal experiments were ethically approved by the Animal Experiment Center of Hunan University of Chinese Medicine (HNUCM) Ethics Committee (Approval number: LL2022091403). All experiments were performed in compliance with national and institutional laws. The acquisition and description of data followed the recommendations are reported in accordance with the ARRIVE guidelines. All animals were kept in the barrier system at the animal experiment center of HNUCM at a controlled temperature and humidity. The rats were randomly categorized into different treatment groups. All surgeries and follow-up analyses were performed through a blinded intervention approach.
Cell isolation, culture, and exosome extraction
First, Sprague–Dawley (SD) rats weighing 100–120 g were euthanized by CO2 inhalation and immersed in 75% alcohol for 15 min to achieve disinfection in preparation for EPCs extraction. Following the manufacturer’s instructions, the EPCs isolation kit was used to extract EPCs, which were then cultured in rat bone marrow-derived endothelial progenitor cells complete medium in a 37 °C and 5% CO2 incubator. After cell passaging, the medium was changed to endothelial cell growth medium-2 (EGM-2). For exosome extraction, 70–80% confluent EPCs were washed with phosphate-buffered saline (PBS) and changed to a fresh exosome-depleted culture medium. After culturing for 24 h, the medium was collected in centrifuge tubes. The medium containing exosomes was extracted through high-speed centrifugation combined with ultrafiltration. The supernatant was subjected to gradient centrifugation at 4 °C: 500×g for 25 min, 3000×g for 15 min, and 12,000×g for 30 min, aiming to remove cellular debris, apoptotic vesicles, and macrovesicles. The cell-free supernatant, cleared of impurities, was then transferred into a 30kD ultrafiltration tube and centrifuged at 3000×g for 25 min to collect exosomes. The resulting precipitate was further purified through centrifugation for 90 min at 120,000×g. Thereafter, the precipitate was resuspended in 200 μL PBS and stored at − 80 °C for further analysis.
VECs were extracted and identified following the methods outlined in previous research40, then cultured in Dulbecco’s modified eagle’s medium/nutrient mixture F-12 (DMEM/F12) medium [with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin] in a 37 °C and 5% CO2 incubator.
Establishment of carotid BI model
15 Male SD rats [Special Pathogen-Free (SPF) weighing 300–350 g; aged 6–8 weeks] were purchased from Hunan Slaughter Jingda Laboratory Co.S, Ltd. (Animal license number: SCXK (Xiang) 2019–0004). High-fat chow was purchased from Beijing Keo Collaborative Feed, and the formulation was prepared as reported in our previous study41. The animals were fed with high-fat chow for two weeks. The experimental method of establishing carotid BI model was described previously42. The rats were anesthetized with 2.5% pentobarbital and injected with penicillin three days post-surgery to prevent infection. The rats in the sham group had only their carotid arteries isolated. All rats with established carotid BI were simultaneously randomized into the BI model group and the EPC-Exo group. The exosome group animals were injected with EPC-Exo (30 μg) 12 h post-surgery and on the third day after the operation, while the other groups were injected with an equal volume of PBS. The rats were euthanized by CO2 inhalation after 14 days. Half of the carotid arteries were fixed in 4% paraformaldehyde and the other half were stored at − 80 °C.
Delivery of exosomes
To monitor the internalization of EPC-Exo, the purified exosomes were labeled using an exosomal red fluorescent labelling dye (PKH26) kit. The exosome group animals were injected with PKH26-labelled EPC-Exo ( 30 μg) 12 h post-surgery and on the third day after the operation, while the other groups were injected with an equal volume amount of PBS (Fig. 7). Whether EPC-Exo adhered to the injured carotid artery vessel wall after injection and the binding of exosomes were assessed by fluorescence. After seven days of modeling, the rats in the model and EPC-Exo groups were euthanized by CO2 inhalation. The intact carotid arteries were extracted and fixed in paraformaldehyde. Subsequently, the fixed carotid arteries were dehydrated, made transparent, embedded and made into paraffin sections. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The sections were visualized and imaged under a fluorescence microscope, focusing on the intimal area of the carotid artery. Image J software was used to analyze the fluorescence intensity.
Animal and cell models establishment and processing flowchart. Figure created with BioRender (https://biorender.com/).
Flow cytometry
The EPCs were centrifuged two times at 1000 rpm to remove the residual medium and then resuspended in PBS. Subsequently, the cells were incubated with CD34 (1:100) and CD133 (1:100) antibodies and left on ice for 30 min. Following incubation, the cells were centrifuged and resuspended in 200 μl PBS, and the results were analyzed by DxP AthenaTM flow cytometry (Cytek, US).
Transmission electron microscope
The exosome solution was first added dropwise to the copper grid and incubated for 5 min at room temperature (RT) before blotting the excess solution with filter paper. Subsequently, 10 μL of 2.5% glutaraldehyde solution was added dropwise to the copper grid for 10 min and washed with PBS 1–2 times for 3 min each. Following that, the surface was washed once with PBS and allowed to dry at RT. Finally, transmission electron microscopy was used to examine the morphology of the exosomes.
Exosome particle size analysis
To avoid clogging the injection needle, the exosomes were removed and diluted to the appropriate multiple with a PBS gradient. After verifying the accuracy of the particle size and concentration analyzer against set standards, we tested the sample by placing it on the machine and obtained information on exosome particle size and concentration.
Hematoxylin and eosin (H&E) staining
To assess intimal hyperplasia after endovascular injury, the sections were stained with H&E staining, focusing on the endovascular area. The fixed vessels were dehydrated, made transparent, embedded, sectioned, and then stained using hematoxylin and eosin. They were then photographed under the microscope to be sealed and dried. Three fields of view (40×) were randomly selected under the microscope, and the mean value was taken as the measurement. The intramembranous area of the inner outer elastic membranes, lumen and their perimeters were analyzed using Image Pro Plus software 6.0. The Intima-media thickness (IT) and hyperplasia ratio of intima thickness (HRIT) were calculated based on previous studies41. The calculations are as follows: IT = (perimeter of the internal elastic tunica − perimeter of the lumen)/2π; MT = (perimeter of the external elastic tunica − perimeter of the internal elastic tunica)/2π; HRIT = IT/(IT + MT) × 100%.
Immunofluorescence (IF)
To identify the EPCs, cultured EPCs were fixed in 4% paraformaldehyde for 30 min. After washing with PBS, the cells were blocked with 3% bovine serum albumin (BSA) for 60 min. The EPCs were then incubated with primary antibodies CD34 (1:500) and CD133 (1:500) at 4 °C overnight. After incubation, the cells were washed with PBS and then stained for 60 min with a fluorescent secondary antibody (1:500) at RT in the dark. Thereafter, the nuclei were stained with DAPI, and the EPCs were examined by immunofluorescence (IF). EPCs have the ability to take up Dil-LDL and bind fluorescein isothiocyanate (FITC)-UEA-1. Cultured EPCs were added to complete medium containing Dil-LDL and cultured in a 37 °C and 5% CO2 incubator for 4 h. The cells were then fixed in 4% paraformaldehyde for 20 min before adding PBS containing FITC-UEA-1 and allowing the mixture to settle at RT for 2 h. Subsequently, the EPCs were stained with DAPI for 6 min. The cells were then fixed in 4% paraformaldehyde for 20 min before adding PBS containing FITC-UEA-1 and allowing the mixture to settle at RT for 2 h. Subsequently, the EPCs were stained with DAPI for 6 min.
To investigate the expression of apoptosis-related proteins in different groups of VECs, the VECs were fixed with 4% paraformaldehyde for 30 min for IF testing. Cultured VECs were blocked with 3% bovine serum albumin (BSA) for 60 min, and then incubated with Cleaved-caspase-3 primary antibody at 4 °C overnight. Thereafter, the VECs were washed and incubated with FITC-conjugated secondary antibody for 1 h. Nuclei were stained with DAPI. All cells were sealed with an anti-fluorescence quenching sealer and photographed under a fluorescence microscope. Image J software was used to analyze the fluorescence intensity.
Immunohistochemistry
Paraffin sections were first dewaxed with water and then antigenically repaired through dropwise addition of an Antigen Repair Solution. Subsequently, the sections were incubated in a 3% hydrogen peroxide solution for 25 min at RT to block the endogenous peroxidase. The sections were then incubated overnight at 4 °C in a wet box with a drop of 3% BSA at RT for 30 min. Primary antibodies [VCAM-1 (1:100)] were then added to the sections. After incubation, the cells were washed before adding a freshly prepared diaminobenzidine (DAB) coloring solution dropwise onto the sections under the microscope for a controlled period. The hematoxylin-stained nuclei were sealed with neutral resin. Three fields of view (40×) were randomly selected under the microscope, and the mean value was taken as the measurement. Pictures were captured under a microscope after drying the sections. Image J software was used to analyze the positive area: Mean density = sum of integral optical density (IOD)/area of measurement area.
Enzyme‑linked immunosorbent assay (EILISA)
The rat serum was obtained and left to stand at 4 °C for two hours, then centrifuged at 3000 rpm for 15 min. The serum was divided and stored at − 80 °C. According to the instructions of the ELISA kit, the levels of ET-1, IL-6, and TNF-α in rat serum was determined.
Cell treatment and proliferation
The cell counting kit-8 (CCK-8) assay was used to detect VECs viability. The VECs were seeded in 96-well plates at a density of 1 × 105 cells/ml. The VECs in the control group were treated with a complete ordinary medium for 24 h, whereas cells in other groups were treated with LPS (1 mg/ml) for 24 h, as outlined in previous research43, to establish an endothelial cell injury model. Furthermore, the control and LPS groups were treated by replacing their culture media with fresh culture media, while the exosome group was treated with a complete culture medium containing exosomes for 24 h (Fig. 7). According to the treatment of cells, the cells were divided into three groups: control (added to complete medium), LPS [both added to complete medium containing LPS (1 μg/ml)], and LPS + EPC-Exo [added to a complete medium containing LPS (1 μg/ml) and complete medium containing EPC-Exo (10 μg/ml)]. Subsequently, a 10% CCK-8 solution was added, and the mixture was placed in the incubator before measuring the absorbance values at a wavelength of 420 nm with an enzyme marker for 1–2 h.
Scratch experiment
VECs were inoculated into 6-well plates (2 × 105 cells/well). After the cells had adhered, a straight line was drawn vertically and evenly in the middle of the well plate with the tip of a gun. The cells were washed with PBS to remove the scratched cells, the control group was treated with regular medium, while the other groups were treated with normal medium containing LPS (1 μg/ml) for 24 h. The cells were incubated at 37 °C in a 5% CO2 incubator. The same location was photographed at 0 h and 24 h. The scratch experiment were observed using an Aiovert A1 inverted microscope (Zeiss, Germany). Respectively, the area before and after injury was analyzed by ImageJ.
Tube formation assay
The matrigel solution was allowed to melt overnight at 4 °C, and the 96-well plates and 200 μl tips were pre-cooled at − 20 °C. The thawed matrigel solution was then added to the 96-well plates on the following day and placed in the incubator for 30 min. Subsequently, 100 μl of cells (1 × 105 cells/ml) were added to the wells. The cell tube formations were observed using an Aiovert A1 inverted microscope (Zeiss, Germany), and three randomly selected fields of view were photographed and counted.
Transferase dUTP nick end labeling (TUNEL) staining
The cells were treated as previously described. Following VECs fixation, PBS containing 0.3% Triton X-100 was added, and the mixture was incubated for 5 min at RT. The cells were then washed two times with PBS after incubation. The TUNEL assay was performed using a TUNEL assay kit per the manufacturer’s instructions. After preparation, 50 μl of the TUNEL assay solution was added to the samples and incubated for 60 min at 37 °C. During cultivation, an appropriate amount of water should be added to the excess well space to keep it moist, thereby minimizing the TUNEL determination solution’s evaporation. After washing, the nuclei were stained with DAPI for 6 min before sealing with an anti-fluorescence quenching blocking solution and observing under a fluorescence microscope.
Actin cytoskeleton staining
Actin cytoskeleton staining using phalloidin is commonly used to study the morphology and integrity of the network of fibers of the cells, which causes the skeleton fiber filaments to exhibit red fluorescence. Cell treatment was performed as previously described. The VECs were fixed and blocked with a 3% BSA solution for 30 min. After washing with PBS three times, 250 μl Phalloidin (1:500) was added and incubated for 1 h at RT. The cells were then stained with DAPI for 6 min. Subsequently, the cells were sealed with an anti-fluorescence quencher after washing three times with PBS and observed under a fluorescent microscope.
Fluorescent quantitative PCR (qPCR)
Following the standard protocols, the total RNA kit was used to extract total RNA from carotid tissues and VECs. The RNA was reverse transcribed to synthesize cDNA and then amplified by PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to evaluate and standardize the relative gene expressions through the 2 − ΔΔCt method. Table 1 shows the primer sequences used.
Western blot (WB) analysis
Rat carotid arteries were transferred into a grinding tube and finely ground before lysing on ice for 30 min to extract the supernatant. Subsequently, Sodium Dodecyl Sulphate (SDS) was added to determine and denature the proteins. Cell and exosome proteins were extracted as previously described without grinding. The proteins were then separated through polyacrylamide gel electrophoresis, transferred to a PVDF membrane, blocked with 10% milk for 1 h, and incubated overnight at 4 °C with primary antibodies CD63 (1:2000), tumor susceptibility gene 101 protein (TSG101) (1:1000), CD81 (1:1 000), Calnexin (1:1000), eNOS (1:500), Bcl2 (1:2000), Bax (1:2000), β actin (1:8000). After washing 3 times with TBS-Tween (TBST), secondary antibodies (1:10,000) were incubated at room temperature for 1 h. Bands were detected by GelDocXR + Gel Imaging System (BioRad, USA) and analyzed by Image J software.
Statistical analysis
All statistical analyses were performed using SPSS software. The data were quantitative and continuous, and the statistics for each test were expressed as mean ± standard deviation (M ± SD). One-way analysis of variance (ANOVA) and the least significant difference (LSD) test were used to compare groups if the normality and homogeneity of variance were met. On the other hand, the Dunnet T3 test was employed when variance was not homogeneous.
- SEO Powered Content & PR Distribution. Get Amplified Today.
- PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
- PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
- PlatoESG. Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
- PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
- Source: https://www.nature.com/articles/s41598-024-55100-x