The Experimental Animal Center of Binzhou Medical University (Yantai, China) provided 36 3-week-old male Sprague Dawley (SD) rats weighing 80-100 g. The animals were housed in standard cages and maintained under standard conditions at constant room temperature of 20-25˚C, 40-70% humidity and 12/12-hlight/dark cycle, with unrestricted access to food and water. For anesthesia, 3% pentobarbital sodium (50 mg/kg body weight) was administered intraperitoneally, and SD rats were sacrificed using CO₂ inhalation, with a fill rate of 60% of the chamber volume/min. Death was confirmed using lack of pulse, breathing, corneal reflex, response to a firm toe pinch and graying of the mucous membranes, which conformed to the Action against Medical Accidents Guidelines for the Euthanasia of Animals: 2020 Edition (17). After SD rats were sacrificed, 75% alcohol was used to disinfect them. Under aseptic conditions, bilateral femurs and the thoracic aorta of rats were removed and isolated. The present study was approved by the Animal Ethics Committee of Binzhou Medical University (approval no. 2020105;10 December 2020).

Cell-adhesive screening was used to isolate and culture BMSCs. The bone marrow of the femur of 6 SD rats was rinsed with L-DMED culture medium (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.) using a 5 ml syringe, collected in a sterile centrifuge tube and centrifuged in 1,000 x g for 5 min at 4˚C. The supernatant was discarded and L-DMED was added to the centrifuge tube with 15% fetal bovine serum (FBS; cat. no. 11011-8611; Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.) and cells were resuspended. The cell concentration was adjusted to 4x10⁵/ml and transferred into a culture flask. The culture medium was changed after 2 days incubation at 37˚C with 5% CO₂, cells in suspension were discarded and the culture was continued for further 2 days. In the following 6 days, the medium was changed every 3 days. Each day, an inverted phase contrast microscope (magnification, x200; Olympus Corporation) was used to observe the proliferation and morphology of the cells. The rapid proliferation of cells occurred due to adhesion to the petri dish bottom. After reaching 70-80% confluence, cells were sub-cultured. Flow cytometry assay (NovoCyte, Agilent; software version no. FlowJo 10.8.1, BD company) was performed to analyze the cells after they were sub-cultured for 2-3 generations. Cells were collected, washed in cold PBS, centrifuged in 1,500 x g for 5 min at 4˚C and re-suspended in cold PBS. Flow cytometry was used to analyze cells that were labeled with CD105, CD44, and CD34 antibodies (all 1:1,000; cat. nos. A02997-2, A00052 and A00885-1, respectively; all Boster Biological Technology).

Under aseptic conditions, the adventitia of the thoracic aorta of the rats was isolated from the media and intima. The adventitia was then cut into 0.5-1 mm thick tissue pieces and cultured with complete medium supplemented with 15% FBS. The primary culture of AFs adopted the tissue block adherent culture method, in which pieces were stored in a petri dish and cultivated in an incubator for 2-3 days at 37˚C. After being detached from the tissue block, the cells started dividing and proliferating. For 6-7 days, the cells were added to the bottom of the flask, digested and sub-cultured using 0.08% trypsinase (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.), and the culture medium was changed every other day. AFs at generation 2-3 and 70-80% confluence were sub-cultured in 6-well plates filled with culture medium supplemented with 10% FBS at a density of 1x10⁴ cells/cm². After washing three times with PBS, fixing with 4% paraformaldehyde at room temperature for 15 min, immunocytochemical staining was performed to identify the AFs using 1:100 rabbit anti-Vimentin primary antibody (cat. no. PB9359, Boster Biological Technology). The cells in the plate were washed in PBS, and endogenous peroxidase activities were blocked by 3% H₂O₂ at room temperature for 15 min. After washing with PBS three times, the plates were blocked with goat serum at room temperature for 20 min. Subsequently, the liquid was discarded and the plate dried. The cells were incubated over night at 4˚C with the primary antibody followed by incubation with FITC-conjugated goat anti-rabbit secondary antibody (1:3,000; cat. no. A0562, Beyotime Institute of Biotechnology) for 30 min at 37˚C. The immunofluorescence was observed using a laser confocal microscope (magnification, x200; FV3000; Olympus Corporation).

A vascular injury model was simulated in vitro and BMSCs at generation 2-3 were co-cultured with the supernatant of the AFs culture medium. L-DMED supplemented with 10% FBS was used as a complete culture medium and maintained at 37˚C with 5% CO₂. These conditions were used for all incubations. In Model 1, AFs were cultured for 2 days in complete culture medium supplemented with NE (cat. no. N7960; Beijing Solarbio Biological Technology) at a final concentration of 10 ng/ml. Subsequently, the medium was replaced with a fresh complete culture medium without NE. Cells were cultured for 1 day and supernatant of the culture medium was collected. BMSCs were cultured with supernatant for 6 days and the AFs supernatant was changed every other day. In Model 2, AFs were cultured for 2 days in complete culture medium without NE, followed by 1 day in fresh complete culture medium and supernatant collection. BMSCs were cultured with the AFs supernatant for 6 days and the supernatant was changed every other day. In the control group, BMSCs were cultured for 6 days in complete culture medium without the supernatant of AFs. The complete culture medium was changed every other day. An inverted optical light microscope (magnification, x200) was used to observe the morphological changes of BMSCs cultured in AFs supernatant for 6 days. BMSCs from the three groups were collected and washed with PBS to be used in subsequent experiments

BMSCs were fixed with 4% paraformaldehyde for 10 min at room temperature. BMSC nuclei were stained with DAPI (0.02 g/l; Santa Cruz Biotechnology, Inc.) overnight at 4˚C and incubated with rabbit anti-α-SMA, TGF-β1 and SMAD3 (1:200; cat. nos. AF0048, AF0297 and AF1501, respectively; Beyotime Institute of Biotechnology) primary antibodies at 37˚C for 60 min, followed by incubation with FITC- or Cy3-conjugated goat anti-rabbit IgG (1:300; cat. no. A 0562, A0516; Beyotime Institute of Biotechnology) secondary antibodies at 37˚C for 60 min. Stained cells were observed under a laser confocal microscope (magnification, x200; FV3000; Olympus Corporation).

Total protein was extracted from BMSCs using ice-cold RIPA lysis buffer supplemented with 1 mM phenyl methane sulfonyl fluoride (Beyotime Institute of Biotechnology). Total protein was quantified using the Enhanced BCA Protein Assay kit (cat. no. P0006; Beyotime Institute of Biotechnology) and 40 µg protein/lane was separated by SDS-PAGE on 8-12% gel. The separated proteins were transferred onto a polyvinylidene fluoride membrane (EMD Millipore; Millipore Sigma) and blocked with 5% skimmed milk for 15 min at room temperature. Membranes were incubated with rabbit anti-α-SMA, anti-TGF-β1, anti-SMAD3 and GAPDH (1:1,000; cat. nos. AF0048, AF0297, AF1501 and AF1186, respectively; Beyotime Institute of Biotechnology) primary antibodies at 4˚C overnight. Following primary incubation, membranes were incubated with the horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3,000; cat. no. SA00001-2, Proteintech Group, Inc.) secondary antibody at room temperature for 2 h. The bands were visualized using Millipore Immobilon ECL (Cat. No. WBKLS0100, Millipore Sigma). Protein expression was quantified using Scion Image (version 1.8.0; Scion Corporation) with GAPDH as the loading control.

BMSCs in the logarithmic growth phase were seeded into 96-well plates at a density of 5x10³ cells/well (100 µl/well). The cells were cultured at 37˚C with 5% CO₂ for 12, 24, 36 and 48 h to ensure that they adhered to the plate wall. Cell Counting Kit-8 (CCK-8) reagent (10 µl; cat. no. c0037, Beyotime Institute of Biotechnology) was added to each well and incubated for 4 h. A Bio-Tek microplate reader (TecanGroup, Ltd.) was used to measure the absorbance at 450 nm for the determination of BMSC proliferation.

Transwell plates with 8 µm pore membranes were used to investigate the BMSC migration. In Model 1, AFs were cultured in complete culture medium with 10 ng/ml NE for 2 days, whereas in Model 2 the AFs were cultured in the complete culture medium without NE for 2 days as afore mentioned The AFs in Model 1 and 2 were then centrifuged in 1,500 x g for 5 min at 4˚C and rinsed with PBS solution and the AFs were added separately into the lower chamber of the Transwell plate, while the upper chamber was seeded with 2x10⁵ cells/cm². For the control group, BMSCs (2x10⁵ cells/cm²) were cultured in the upper chamber without AFs in the lower chamber. BMSCs in the three groups were cultured in the complete culture medium at 37˚C with 5% CO₂ for 6 days to observe morphological changes, proliferation and migration under an inverted microscope. The membrane was collected and the cells on the upper surface of the membrane were removed with cotton swabs. The membrane was washed with PBS and cells adhering to the membrane were fixed with 4% paraformaldehyde at room temperature for 15 min. BMSC nuclei were stained with DAPI (0.02 mg/ml; cat. no. c1002; Beyotime Institute of Biotechnology) overnight at 4˚C and rinsed five times with PBS to wash away the unbound DAPI. Stained cells were observed in five randomly selected fields of view under laser confocal microscopy (magnification, x100) to measure the migration of BMSCs.

Data are presented as the mean ± standard error of six independent experimental repeats. The significance of the differences was examined by one-way ANOVA followed by Duncan's multiple range tests, Statistical analysis was performed using SPSS20.0 (IBM Corp.) and GraphPad Prism 6 (GraphPad Software; Dotmatics000.0). P<0.05 was considered to indicate a statistically significant difference.

By using the cell adherent screening method, BMSCs were isolated, cultured and observed under an inverted microscope. After 3 days of culture, round monocytes adhered to the wall, whereas hematopoietic stem cells did not adhere to the wall, allowing them to be discarded in the exchange fluid of culture medium. The primary culture of BMSCs showed a short shape and mostly round and unextended. A few extended cells started to appear and proliferate at 3-5 days. Cells were typically spindle-shaped with 2-3 long protrusions, while a few cells were polygonal and rod-shaped with large oblate nuclei and 1-2 nucleoli (Fig. 1A). Cells were sub-cultured after 5-6 days of cell culture. In cell culture, cell morphology gradually homogenized into long spindle shape, cells proliferated rapidly and exhibited swirls (Fig. 1B). As soon as 2-3 generations of BMSCs were obtained, flow cytometry analysis revealed that the BMSCs were negative for CD34, while they were positive for CD44 and CD105 (Fig. 1C). According to these findings, cultured cells had morphological and immunophenotypical characteristics similar to those of BMSCs.

As observed under an inverted microscope, AFs grew from the edge of the vascular adventitial tissue block and divided 2-3 days later. The primary cultured AFs also demonstrated adherent growth with large and oblate nuclei (Fig. 2A). Under the microscope, the adherent cells were irregular polygons or fusiform, growing vigorously and overlapping in crista-like layers (Fig. 2B). Fluorescence staining indicated that AFs cells were positive for vimentin expression (Fig. 2C).

Induction and differentiation of BMSCs were performed using cells at generations 2-3. An inverted microscope was used to observe the morphology of the cultured cells. After 6 days of BMSCs culture with supernatant of AFs, the BMSCs had exhibited 70-80% confluence in Models 1 and 2. BMSCs morphology had lost the characteristic swirls state, while some cells changed their long spindle shape and exhibited an elliptical appearance. There was no notable change in cell morphology in the control group. A total of three groups of cell slides were prepared for immunofluorescence staining. The cytoplasm expressing α-SMA showed red fluorescence due to the Cy3-conjugated second antibody goat anti-rabbit IgG, while positive expression of TGF-β1 and SMAD3 in the cytoplasm was indicated by a green fluorescence due to the FITC-conjugated secondary goat anti-rabbit IgG. Immunofluorescence showed that after cell culture for 6 days, Model 1 exhibited the strongest staining compared with that in model 2 and the Control group (Fig. 3A).

Western blotting was used to analyze expression of α-SMA and TGF-β1/SMAD3 signaling pathway proteins in the BMSCs (Fig. 3B). Models 1 and 2 differed significantly from the control (P<0.05). Compared with those in the control group, the expression levels of α-SMA, TGF-β1 and SMAD3 in BMSCs in models1 and 2 were increased (P<0.05; Fig. 3C), with model 1 showing the highest increase.

The proliferation of BMSCs was evaluated using the CCK-8 method. BMSCs from Model 1 and 2 were cultured with AFs supernatant for 12, 24, 36 and 48 h, while BMSCs cultured in normal medium were used as the control group (Fig. 3D). There were all have significant differences among the three groups of the proliferation of BMSCs (P<0.05), and the Model 1showing the highest proliferation rate.

According to Transwell migration assay, BMSCs migrated from the upper to the lower chamber through the membrane in all three groups. BMSCs migration in Model 1 was significantly higher than that in Model 2 and Control after 6 days of culture (P<0.05; Fig. 4A and B).