Cell Culture
VSMCs were purchased from the National Platform of Experimental Cell Resources for SciTech (Beijing, China). They were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA) with 10% foetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (P1400, Solarbio, Beijing, China). The culture medium was refreshed every 3 days and the cells were cultured at 37°C with a humidified atmosphere of 5% CO2. To induce calcification, VSMCs were cultured in a medium containing 10 mM β-glycerophosphate (β-GP; 50020, Sigma-Aldrich, St. Louis, MO, USA) to induce the osteoblastic differentiation of VSMCs. To reveal the effect of exosomes isolated from mice subjected to room temperature exposure (RT-Exo) or CT-Exo on the osteoblastic differentiation of VSMCs and the mechanism involved, VSMCs were incubated with 200 ng/μL of CT-Exo or RT-Exo in the subsequent experiments.
To investigate the effect of autophagy on VSMC calcification, cells were pre-treated with 5 mM of the autophagy inhibitor 3-MA (5142-23-4; SelleckChemm, USA) or 1 µM of the autophagy inducer RAPA (53123-88-9; SelleckChem) for 30 min. The cells were treated with β-GP for various times and then collected for different experiments: after 3 days, cells were collected for western blotting; after 10 days, cells were collected for senescence-associated β-galactosidase (SA-β-gal) staining (C0602; Beyotime Institute of Biotechnology, Shanghai, China); after 14 days, cells were collected for alkaline phosphatase (ALP) activity detection (A059-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and ALP staining (Solarbio); and after 28 days, cells were collected for ARS staining (G1038; Servicebio, Wuhan, China).
Agonists and inhibitors of the AMPK/mTOR signalling pathway were used to investigate its role in calcification. VSMCs were stimulated with 10 µM of Compound C (S7306; SelleckChem) or 10 µM of MHY1485 (S7811; SelleckChem) for 30 min and then treated with 200 ng/μL of CT-Exo for 48 h. p-AMPK, t-AMPK, p-mTOR, t-mTOR and RUNX2 protein expression was evaluated in the cell lysates. The SA-β-gal and ARS staining was the same as described above; CT-Exo, Compound C and MHY1485 were changed once every 3 days for a period of 10 or 28 days, respectively.
Plasma Collection and Administration
Six-week-old male mice (n = 6) were systemically treated with phosphate-buffered saline (PBS), CT plasma or CT-Exofree plasma (100 μL/injection) via tail intravenous injection 8 times over 24 days[36]. CT plasma was isolated from mice subjected to CT for 30 days (4–8°C) Two weeks later, the mice were intraperitoneally injected with vitamin D (VD) for 5 days. CT-Exofree plasma was produced as follows: CT plasma was diluted with PBS (1:4, v/v), and then ultracentrifuged at 100,000 g for 18 h to collect the supernatant. After centrifugation, the exosomes were concentrated at the bottom of the test tube and about 80% of the upper plasma had been collected, CT-Exofree plasma was filtered by 0.22 μm filter and centrifuged at 4,000 g to approximately the initial plasma volume by ultrafiltration in a 15 mL Amicon Ultra-15 centrifugal filter unit (Millipore, Billerica, MA, USA). The exosomes were stored at –80°C before use.
Isolation and Characterisation of Exosomes
Plasma samples were obtained from RT mice (kept at 22–25°C) or CT mice (kept at 4–8°C) for 30 days. Briefly, the whole blood was collected into eppendorf (EP) tubes containing Ethylene Diamine Tetraacetie Acid (EDTA) anticoagulant. Blood samples were processed within 30 min of collection. The mixture was centrifuged to collect the plasma at 3,000 g for 20 min. Subsequently, the plasma underwent successive centrifugation at 3,000 g for 20 min and then 10,000 g for 30 min to discard dead cells and cellular debris. The final supernatant was ultracentrifuged at 100,000 g for 120 min. The supernatant was removed and resuspended in PBS, ultracentrifuged again at 100,000 g for 120 min (avoiding freeze-thaw cycles) and then re-suspended in 15 mL of PBS. The suspension was filtered through a 0.22 μm filter steriliser (Millipore) and centrifuged at 4,000 g to approximately 200 μL by ultrafiltration in a 15 mL Amicon Ultra-15 centrifugal filter unit (Millipore). All procedures were performed at 4°C. Exosomes were stored at −80°C or used for the downstream experiments.
The exosomal protein content was quantified with the BCA protein assay kit (P0012; Beyotime). Transmission electron microscopy (TEM; H-7650, Hitachi, Tokyo, Japan) and dynamic light scattering (DLS) with a Nanosizer™ instrument (Malvern Instruments, Malvern, UK) were used to observe the morphology and measure the size distribution of exosomes, respectively. The protein expression of exosomal markers (TSG101, CD81 and CD9) was assessed by western blotting.
For in vitro assays, exosomes in different groups were used at the concentration of 200 ng/μL. For in vivo experiments, exosomes were used at 200 μg (dissolved in 100 μL PBS for intravenous injection) per time and per mouse.
TEM
VSMCs were fixed overnight in 2.5% glutaraldehyde and post-fixed in 1% osmic acid for 2 h. The samples were then dehydrated, embedded and sectioned. After being double stained with 3% uranyl acetate and lead nitrate, the autophagic structures in the cells were viewed using a TEM (H-7650, Hitachi, Tokyo, Japan).
Exosome Uptake Assay and Tracing
In vitro, CT-Exo were labelled with PKH26 red fluorescent dye (MINI26-1KT, Sigma-Aldrich) following the manufacturer’s protocol. After removing the unbound dye, CT-Exo were added to the VSMCs and incubated at 37°C for 6 h. After discarding the culture supernatant and washing the cells with PBS, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min and then incubated with DAPI (C0065; Solarbio) to stain the nuclei. The uptake of the red PKH26-labeled CT-Exo by VSMCs was determined with a fluorescence microscope (Nikon Instruments Korea, Seoul, Korea).
In vivo, to explore whether CT-Exo could be transported from bone to blood vessel walls after intramedullary injection, 100 μL of 1 μg/μL CT-Exo were labelled with 5 μL of 200 μg/mL 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR; 2024243, Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Then, the same was ultracentrifuged to remove unbound dye. Mice were injected with DiR-labelled CT-Exo via the tail vein injection for 3 consecutive days. Live imaging was performed 24 h after the last injection. The mice were killed, organs were removed for photographing, the thoracic aorta of the mice was dissected and immunofluorescence staining was performed on quick frozen sections to analyse the uptake of exosomes in arterial vessels. An anti-TSG101 antibody (1:250, bs-1365R, Bioss, Beijing, China) was used to label exosomes.
Measurement of Reactive Oxygen Species (ROS) Generation
Intracellular ROS production was measured by flow cytometry using the cell-permeable fluorogenic probe DCFH-DA (S0033S; Beyotime) according to the manufacturer’s instructions. Briefly, calcified VSMCs were treated with 200 ng/µL of CT-Exo or PBS for 6 days, washed three times with PBS and then incubated with 1 × 10−5 µM DCFH-DA at 37°C for 20 min.
Apoptosis Assay
VSMCs were treated with CT-Exo or PBS with or without β-GP for 3 days. Apoptosis was measured using the Annexin V-FITC/PI Detection Kit (556547, BD Bioscience, USA) according to the manufacturer’s protocol. For Annexin V-FITC/PI staining, the treated cells were harvested, washed twice with PBS and resuspended in 300 µL of 1× binding buffer, at room temperature in the dark, followed by incubation with 5 µL of Annexin V-FITC for 15 min and 10 µL of PI solution for 5 min. Next, the cell suspension was diluted with 200 µL of annexin V binding buffer and analysed by flow cytometry.
Animal Study
Mice were housed in the Animal House of the Second Xiang-Ya Hospital with a 12-h photoperiod. Male mice were injected intraperitoneally with VD (500 U/g/day) for 5 days to induce arterial calcification and ageing. Mice were fed with regular chow throughout the entire experiments. The RT mice were kept at 22–25°C for 30 days. The CT mice were first kept at 18°C for 7 days (for adaptation), and then kept at 4–8°C for another 30 days. The 4–8°C cold room was equipped with a ventilation system that allowed cold air to circulate.
After 30 days of RT or CT, the mice were administered a high-dose of VD for 5 consecutive days, followed by waiting for 7 days. This treatment occurred at either RT or CT, depending on the initial 30-day treatment. All live mice (n = 6) were sacrificed via intraperitoneal injection of sodium pentobarbital (50 mg/kg) followed by cervical dislocation. The thoracic aorta was embedded in paraffin, sectioned and then stained with ARS. The artery from the aortic arch to the iliac branch was isolated for determination of arterial wall calcium content. No mice died during the experiment.
The impact of CT-Exo and RT-Exo on acute arterial calcification and the role of miR-320a-3p in the CT-Exo-induced alleviation of arterial calcification were also evaluated. Mice were injected intravenously with 200 μg of CT-Exo, AntagomiR-320a-3p or AntagomiR-NC-pre-treated CT-Exo, or an equal volume of PBS (100 μL per mice) every 3 days until the end of the experiment (n = 6 per group). At the same time, the mice were injected with VD for 5 consecutive days, followed by waiting for 7 days. Blood samples were collected to detect the levels of blood urea nitrogen (BUN), creatinine (CREA), calcium, and phosphorus using an automatic biochemical analyser (Chemray 800; Redu Life Technology, Shenzhen, China). The thoracic aortas were dissected. Immunohistochemistry was carried out to determine the levels of RUNX2 in aortic tissues. ARS or Von Kossa staining (G1043; Servicebio) was used to detect artery calcification. Finally, the calcium content was measured.
Next, whether CT-Exo exert an inhibitory effect on MAC in vivo through the autophagy pathway was investigated. The mice were randomly divided into six groups (n = 6 per group): PBS (CTRL), VD+PBS (PS), VD+CT-Exo (CT-Exo), VD+3-MA (3-MA), VD+RAPA (RAPA) and VD+CT-Exo+3-MA (CT-Exo+3-MA). Mice were intraperitoneally injected with either 3-MA (15 mg/kg) or RAPA (2mg/kg) starting 5 days before the first CT-Exo injection (CT-Exo was injected every 3 days for a total of eight injections) until the experiment was terminated. Then, arterial calcification was induced by VD 2 weeks before the mice were sacrificed. One mouse from the CT-Exo+3-MA group and the RAPA group died from unknown causes after being treated four times. Immunohistochemistry was carried out to determine p21 expression in aortic tissues. MAC was detected by ARS and Von Kossa staining and the calcium content was measured.
In another experiment, CT mice were injected intraperitoneally with GW4869 (2 mg/kg; S7609, SelleckChem) to inhibit circulating exosomes[37, 38]. Immunohistochemistry was carried out to determine RUNX2 expression in aortic tissues. ARS staining were used to detect MAC. Finally, the calcium content was measured.
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated from cells with TRIzol Reagent (Invitrogen) based on the manufacturer’s instructions[39]. For miRNA detection, miRNA was reverse transcribed and analysed by TB Green® Premix Ex Taq™ II (Tli RNaseH Plus; RR820A, Takara, Kyoto, Japan) based on the manufacturer’s protocol and using U6 as the normalisation control. U6 (HmiRQP9001) and miR-320a-3p (HmiRQP0405) primers were purchased from GeneCopoeia (Guangzhou, China).
RNA Sequencing
The RT-Exo and CT-Exo groups were selected for RNA sequencing (n = 3 per group). Total RNA was extracted and quantified using a NanoDrop spectrophotometer and an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA). A messenger RNA (mRNA) library was then constructed and amplified with Phi29 to produce 100 base pair reads on the BGIseq500 platform (BGI, Shenzhen, China). SOAPnuke (V1.5.2) was used to filter the sequencing data, and Bowtie2 (V2.2.5) was used to compare the clean reads with the gene database established by Shenzhen Beijing Genomics Institute to calculate gene expression levels and identify differentially expressed genes (DEGs) (fold-change > 1.5, q < 0.05). The annotated DEGs were analysed using Phyper based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Gene set enrichment analysis (GSEA) was used to evaluate DEGs enriched for either negatively or positively correlated genes.
RNA Interference
Small interfering RNAs (siRNAs) and the negative control RNA duplex (siRNA-NC) were purchased from GenePharma Biotech (Shanghai, China). The miR-320a-3p mimics or miR-320a-3p inhibitor and scrambled oligonucleotides (mimics NC or inhibitor-NC) were purchased from GenePharma Biotech. These were transfected into cells during the logarithmic growth phase. The transfection was performed using the GP-transfect-Mate transfection reagent (GenePharma Biotech) according to the manufacturer’s protocol. The transfected sequences of the miR-320a-3p mimics/inhibitor and siRNA oligonucleotides are shown in Additional file 1, Table S1.
AntagomiRs were purchased from GenePharma Biotech. CT-Exo were transfected with AntagomiR-320a-3p or AntagomiR-NC at 200 nM for 60 min at 37°C. The AgomiRs and AntagomiRs that were not transfected were removed by centrifugation at 4,000 g for 5 min using a 100 kDa Amicon Ultra-4 Centrifugal Filter Unit (Millipore)[20]. The internalisation of AntagomiR-NC-Cy3 by CT-Exo was assessed by qRT-PCR. Treatment with CT-Exo and other AntagomiRs was used for subsequent experiments.
Western Blotting
Total protein was extracted from cultured VSMCs, artery samples or exosomes with radioimmunoprecipitation assay (RIPA) buffer (P0013B; Beyotime). The protein concentration was measured by the BCA assay. Total protein (20–40 µg) was submitted to 8%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation. The separated protein was transferred onto 0.2 or 0.45 µm polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were incubated in 5% non-fat milk or bovine serum albumin (BSA) (depending on the primary antibody), followed by incubation overnight with primary antibody. The following primary antibodies were used: anti-CD9 (ab92726, Abcam, 1:2000), anti-CD81 (ab109201, Abcam, 1:1000), anti-TSG101 (bs-1365R, Bioss, 1:500), anti-RUNX2 (ab76956, Abcam, 1:1000), anti-BMP2 (bs-10696R, Bioss, 1:500), anti-p53 (10442-1-AP, Proteintech, 1:3000), anti-p62 (18420-1-AP, Proteintech, 1:2000), anti-ATG5 (66744-1-Ig, Proteintech, 1:4000), anti-LC3B (14600-1-AP, Proteintech, 1:4000, to determine the LC3B-II:LC3B-I ratio), anti-PDCD4 (12587-1-AP, Proteintech, 1:1000), anti-p-AMPK (sc33524, Santa Cruz, 1:500), anti-t-AMPK (sc25792, Santa Cruz, 1:500), anti-p-mTOR (2971, CST, 1:1000), anti-t-mTOR (2983, CST, 1:1000), anti-β-actin (20536-1-AP, Proteintech, 1:3000) and anti-GAPDH (10494-1-AP, Proteintech, 1:5000). After washing the blots, they were incubated in secondary antibody conjugated to horseradish peroxidase (SA00001-1 or SA00001-2, Proteintech, 1:5000) for 1 h at room temperature. The immunoreactive bands were visualized with chemiluminescent assay using a chemiluminescence kit (RPN2232, Amersham Biosciences Ltd., UK) and then analysed with an Amersham Imager 600 (General Electric, USA) and Image-Pro Plus software (version 6.0). The relative protein expression level was normalised to the intensity of the β-actin or GAPDH band.
Luciferase Reporter Assay
For the luciferase reporter assay, VSMCs were co-transfected with a luciferase reporter carrying the wild-type PDCD4 3′-untranslated region (UTR), a mutant PDCD4 3′-UTR and miR-320a-3p mimics or scramble oligonucleotides. Forty-eight hours after transfection, luciferase activity was quantified with the luciferase assay system (Promega, Madison, WI, USA). The nucleotide sequences of primers for the construction and mutation of 3′ UTR PDCD4 mRNA were purchased from Ribobio (Guangzhou, China).
Immunohistochemistry
As mentioned above, the expression of RUNX2 and p21 in aortic tissue was examined by immunohistochemistry[38]. In brief, arterial tissue sections were incubated at 65°C for 2 h, dewaxed in turpentine twice for 10 min each; and rehydrated in 99%, 85% and 75% ethanol for 5 min each. Antigen retrieval was performed in a trypsin-EDTA solution. Next, sections were blocked with 5% BSA for 30 min at room temperature and incubated with specific primary antibodies, including anti-RUNX2 (bs-1134R, Bioss, 1:300) and anti-21 (10355-1-AP, Proteintech, 1:400) at 4°C overnight. The following day, sections were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (PV‑9000, ZSGB‑BIO, Beijing, China) at room temperature for 30 min. For control experiments, the primary antibody was replaced by PBS. Finally, the sections were incubated with DAB chromogenic solution (DA1015; Solarbio) for 1 min at room temperature. Nuclei were counterstained with haematoxylin (Solarbio) for 1 min at room temperature. The stained tissue was observed under a CX31 light microscope (Olympus Corporation, Japan). Images were taken at 100× magnification and analysed images using Image-Pro Plus software (version 6.0).
Analysis of Vascular Calcium Content
Arterial samples were decalcified with 0.6 N HCl at 4°C for 48 h. After determining the protein concentration, the calcium content in the supernatant was assessed using a commercial kit (C004-2-1; Nanjing Jiancheng Bioengineering Institute). The vascular calcium content was normalised to the protein concentration.
Statistical Analysis
All data are presented as the mean ± standard deviation of three independent experiments. Data were analysed and plotted using GraphPad Prism software (San Diego, CA, USA) and ImageJ software (National Institutes of Health, Bethesda, MD, USA). The unpaired, two-tailed Student’s t-test was conducted to compare two groups. One- or two-way analysis of variance (ANOVA) with the Bonferroni post hoc test was used to compare three or more groups. Results were considered significant when the p-value was < 0.05. In the Figures, statistical significance is indicated as ns > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.