Study subjects

Thirty patients and thirty healthy individuals were enrolled from October 2023 to January 2024 in this study (Supplementary Table 1). Patients diagnosed with TAK were recruited from the department of rheumatology and clinical immunology in Peking Union Medical College Hospital. All patients were diagnosed according to the American College of Rheumatology criteria 1990 for Takayasu’s arteritis [13]. The disease activity of TAK was defined by the National Institutes of Health (NIH) criteria ≥ 2 points [14]. The study protocol was approved by the Ethics Committee of Peking Union Medical College Hospital (Approval S-478) and conformed to the ethical guidelines of the 1975 Declaration of Helsinki. All individuals involved in this study provided written informed consent prior to enrollment.

Cell culture and transfection

Immortalized human aortic vascular smooth muscle cells (Meisen Chinese Tissue Culture Collections, China) were maintained in smooth muscle cell medium (ScienCell, Carlsbad, USA). The human embryonic kidney epithelial cell line HEK 293T (Shanghai Institute of Cell Biology of Chinese Academy of Sciences, China) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, ThermoFisher Scientific, USA) with 10% fetal bovine serum (Gibco, ThermoFisher Scientific, USA) and 1% penicillin-streptomycin (NCM biotech, Suzhou, China). The cell lines were incubated in 5% CO2 at 37 °C. Phosphate-buffered saline (PBS) used in cell culturing was purchased from Bio-Channel (Bio-Channel, China).

For the transfection process, the VSMC were transfected with miR-199a-5p mimics or a miR-199a-5p inhibitor, as well as corresponding scrambled controls (Sangon Biotech, China) using Lipofectamine™ 2000 (Gibco, ThermoFisher Scientific, Carlsbad, CA, USA) based on the manufacturer’s instructions. VSMC were incubated in 6-well plates and subjected to transfection at 70% confluency. After 24 h of transfection, the media was changed and continued to cultivate for 24 h. The efficiency of mimic and inhibitor transfection was confirmed by real-time polymerase chain reaction.

Rno-miR-199a-5p mimics:

5′-CCCAGUGUUCAGACUACCUGUUC-3′,

5′-ACAGGUAGUCUGAACACUGGGUU-3′.

Mimic negative control:

5′-UUGUACUACACAAAAGUACUG-3′,

5′-GUACUUUUGUGUAGUACAAUU-3′.

Rno-miR-199a-5p inhibitor: 5′-(mG)(mA)(mA)(mC)(mA)(mG)(mG)(mU)(mA)(mG)(mU)(mC)(mU)(mG)(mA)(mA)(mC)(mA)(mC)(mU)(mG)(mG)(mG)-3′.

Inhibitor control: 5′-CAGUACUUUUGUGUAGUACAA-3′.

Enzyme-linked immunosorbent assay (ELISA)

Blood samples were obtained from individuals with TAK and healthy controls utilizing coagulation-promoting tubes. Subsequent to centrifugation at 1800×g for a duration of 10 min, the supernatant was meticulously collected. The derived serum was then conserved at a temperature of -80℃ for future analytical purposes. The concentrations of matrix metalloproteinase-2 (MMP2) and tissue inhibitor of metalloproteinases-2 (TIMP2) within the serum were measured utilizing Human MMP-2 ELISA Kit (MULTI SCIENCES BIOTECH, China) and Human TIMP-2 ELISA Kit (MULTI SCIENCES BIOTECH, China), in strict adherence to the stipulated manufacturer’s protocol. For the quantification of TIMP2, the serum was diluted at a ratio of 1:5 using assay buffer. Absorbance measurements were conducted with a microplate reader (Biotek SynergyNeo2). The ultimate concentrations were deduced from standard curves. Disparities between the cohorts were evaluated employing a paired or unpaired Student’s t-test, depending on the contextual appropriateness of the comparison.

Total exosome isolation

Exosomes present in the serum were extracted utilizing Exosome Isolation and Purification Kit (Umibio, China), following the manufacturer’s instructions. The purified exosomes were from serum validated utilizing transmission electron microscope (TEM) and immunoblotting for markers such as calnexin, CD63, CD9, and TSG101. The dimensions and abundance of the purified exosomes were evaluated by NanoFCM N30E (NanoFCM, China).

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

The total RNA of transfected cells was extracted using RNA simple Total RNA Kit (TIANGEN BIOTECH, China) and quantified by NanoDrop One ultra-micro UV spectrophotometer (ThermoFisher Scientific, USA). The reverse transcription of total RNA into cDNA was performed using PrimeScript™ RT reagent Kit (Perfect Real Time) (Takara Bio, Japan). qRT-PCR was performed on a C1000 Thermal Cycler CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, USA) using BeyoFast™ SYBR Green qPCR Mix (2X) (Beyotime Biotechnology, China). The isolation of miRNA was conducted utilizing miRcute miRNA Isolation Kit (Tiangen, China). The quantitative expression levels of miR-199a-5p were ascertained through the application of qPCR, facilitated by the Hifair® miRNA 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, China) and Hieff® miRNA UniversalqPCR SYBR Master Mix (Yeasen Biotechnology, China). These procedures were executed in strict accordance with the stipulated instructions provided by the manufacturer. The primer sequences used are listed in Supplementary Table 3. The thermocycler conditions were as follows: 37 °C for 30 s; 95 °C for 15 min; followed by 40 cycles of 95 °C for 10 s; 55 °C for 30 s; and 72 °C for 30 s. The relative expression of target genes was calculated by the 2 − ΔΔCq method.

Western blot analysis

The transfected cells at approximately 95% confluence were used for protein extraction. Total proteins were extracted by Radio Immunoprecipitation Assay lysis buffer (Beyotime Biotechnology, Shanghai, China) and separated by 4–20% SDS-PAGE (ACE Biotechnology, Changzhou, China), and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with TBST containing 5% bovine serum albumin (BSA) and then incubated with primary antibodies at 4 ℃ overnight. After incubation with secondary antibodies for 1 h, the PVDF membranes were visualized with an enhanced chemiluminescence system kit (Millipore, Bedford, MA, USA), and the relative protein expression levels were analyzed by ImageJ. The primary antibodies and secondary antibodies used in this study are listed in Supplementary Table 2.

Cell proliferation assays and cell migration assay

The proliferative activity of VSMC was evaluated employing 5-Ethynyl-2′-deoxyuridine (EdU) Apollo Kit (Beyotime Biotechnology, China) or Cell Counting Kit-8 (CCK8; APExBIO, USA). For the EdU assay, the transfected cells were seeded onto a 24-well plate at a density of 1 × 10^5 cells per well. The subsequent procedures were executed in strict accordance with the stipulated instructions provided by the manufacturer. The detection process was facilitated using a fluorescence microscope (IX-73, OLYMPUS, Japan). For the CCK8 assay, the transfected cells were seeded onto a 96-well plate at a cellular density of 1 × 10^3 cells per well, establishing four replicative wells for each experimental group. At designated time points—0 h, 24 h, 48 h, 72 h, and 96 h—10 µl of CCK8 solution was incorporated into each well. Post-incubation for 2 h at 37 °C, absorbance measurements were acquired at a wavelength of 450 nm utilizing a microplate reader (ELX800, BioTek, USA).

The scratch assays and the migration assay were performed to assess cell migration. The transfected VSMC were incubated in a 6-well dish and a scratch lesion was introduced across the confluent cell monolayer using a 10 µL pipette tip. Images of the scratched area were captured at 0 h, 24 h and 48 h utilizing an inverted fluorescence microscope (Olympus IX51, Japan). The migration ability of transfected VSMC was determined using the formula (S0 - St)/ S0, where S0 represents the initial wound area at 0 h, and St corresponds to the wound area at either the 24-hour or 48-hour time point. For the migration assay, a suspension of transfected VSMC was introduced into the upper chamber of a 24-well Transwell plate that contained an 8-µm pored membrane filter (Corning, USA). Following a 48-hour incubation period, cells that had migrated to the lower surface of the Transwell chamber were immobilized using 4% paraformaldehyde for a duration of 30 min. Subsequently, these cells were stained with 0.05% crystal violet for a period of 10 min and subsequently examined under a microscope (Olympus, Japan).

Flow cytometric analysis of apoptosis

The frequency of apoptosis in VSMC was assessed using Annexin V-FITC Apoptosis Detection Kit (Beyotime, China). The transfected VSMC were rinsed twice with PBS. This was followed by the addition of 1× Binding buffer to obtain a cell concentration of 1 × 10^6 cells/ml. Propidium iodide was then added in the absence of light. After an incubation period of 30 min in an ice bath protected from light, VSMC were instantly subjected to flow cytometer (BD FACSCalibur, USA).

Fluorescence in situ hybridization (FISH)

Cells were seeded onto slides placed at the base of a 24-well plate at a density of 6 × 10^4 cells per well. Prior to the experiment, cell density was adjusted to achieve a confluence of 60–70%. FISH assay was conducted to determine the localization of MMP2 and miR-199a-5p within VSMC. In summary, following a prehybridization step at 37 °C for 35 min, the cell climbing pieces underwent overnight hybridization at 37 °C with 2.5 µL 20 µM FAM-labelled miR-199a-5p probes and Cy3-labelled MMP2 probes (RiboBio, China), followed by staining with 4’,6-diamidino-2-phenylindole (DAPI). Images of the slides were captured using confocal laser scanning microscopy (Zeiss, Germany).

RNA immunoprecipitation (RIP) assay

The RIP assays were conducted utilizing the EZ-Magna RNA-binding Protein Immunoprecipitation Kit (Millipore, Germany). Following lysis with RIP buffer, VSMC underwent incubation with magnetic beads in conjugation with anti-AGO2 antibodies or immunoglobulin G (Proteintech Group, China). After isolation from the immunoprecipitated protein-RNA complex, the purified RNA underwent quantified by quantitative polymerase chain reaction (qPCR) to quantify the expression of MMP2.

Dual-luciferase reporter assay

HEK-293 T cells were propagated in 6-well plates a density of 2 × 10^5 cells per well and incubated for a duration of 18 h. Subsequently, HEK-293 T cells underwent co-transfection with MMP2-WT-pmirGLO or MMP2-MUT-pmirGLO reporter plasmids with miR-199a-5p mimics. Dual Luciferase Reporter Gene Assay Kit (Beyotime, China) was employed to measure luciferase activity, with all procedures meticulously adhering to the manufacturer’s prescribed instructions. The relative light units (RLU) of both luciferases were ascertained through biochemical luminescence. Using sea kidney luciferase as an internal reference, the RLU value derived from the firefly luciferase measurement was normalized by dividing it by the RLU value obtained from the sea kidney luciferase measurement. This facilitated the comparison of reporter gene activation levels across different treatments based on the calculated ratios.