Single-Cell RNA-Sequencing and Metabolomics Analyses Reveal the Contribution of Perivascular Adipose Tissue Stem Cells to Vascular Remodeling

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Adipogenic and osteogenic differentiation of PV-ADSCs
Cultured PV-ADSCs were seeded on gelatin coated flasks and then changed to adipogenic or osteogenic differentiation medium and maintained for 2 weeks before being harvested for Q-PCR or subjected to immunofluorescent staining. Differentiation medium was composed of StemXVivo Osteogenic/Adipogenic Base Media (R&D systems, CCM007) with either StemXVivo Adipogenic Supplement (R&D systems, CCM011) or StemXVivo Osteogenic Supplement (R&D systems, CCM009). Immunofluorescent staining of FABP4 (Abcam, ab92501) and OPN (R&D systems, AF808) was utilized to confirm the adipogenic and osteogenic differentiation respectively.

RNA (including miRNA) extraction, RT-PCR and quantitative real-time PCR (Q-PCR)
Total RNA was extracted using the RNeasy mini kit (Qiagen, 74106) following the kit instructions. Reverse transcription of RNA was performed with the QuantiTect Reverse Transcription Kit (Qiagen, 205314). 1000 ng of total RNA was used for each reaction of reverse transcription. Gene expression level in cells was detected by Q-PCR using Eppendorf Mastercycler ep realplex in duplicates. Fold change of gene of interest was calculated by the threshold cycle value difference against internal control GAPDH. Extraction of microRNA was performed with miRNeasy mini kit (Qiagen, 217004) following the kit instructions. Reverse transcription of miRNA was performed with MicroRNA Reverse Transcription Kit (Life Tech,4366597). Specific TaqMan microRNA assay and TaqMan Master Mix (Life Tech, 4440040) were used to assess the expression of microRNAs. All samples were run in duplicates and standardized to U6.

Western blot
10-50 µg of the protein was mixed with 1xSDS loading buffer and then boiled at 95°C for 10 minutes before being loaded to the NuPage 4-12% Bis Tris gel immersed in NuPage MOPS SDS running buffer in a XCell SureLock Mini-Cell (Life Tech, NP0335BOX). Protein ladder (Bio-rad, 161-0374) was loaded simultaneously with the samples and gel was run at 160V for about 75 minutes until the marker with the lowest molecular weight was at the bottom of the gel. Protein was transferred from the gel to the membrane (VWR, 732-3031) in 1X transfer buffer (Life Tech, NP-0006-1) for 45 minutes. The membrane was then blocked with 5% milk in PBS-Tween and incubated with primary antibody diluted in 5% milk at room temperature for 2 hours or at 4°C overnight. Following primary antibodies were used: CNN1 (Abcam, ab46794), TAGLN (Abcam, ab14106), ACTA2 (Sigma, 2547-100), and TGFb1 (Abcam, ab92486). Secondary antibodies were purchased from Dako (Rabbit anti-goat, P0449; Rabbit anti-mouse, P0260; Swine antirabbit, P0217). Further 3 washes were performed before addition of ECL detection solutions (Fisher Scientific, RPN2106). After incubation with the detection solution for 2 minutes, exposure of the films (Fisher Scientific, 28-9068-37) was carried out with the Compact X4 (Xograph Imaging System).

Histological analysis
Tissues including subcutaneous Matrigel plugs and vein grafts from experimental mice were fixed with 4% formalin overnight at 4°C prior to a machine-based dehydration. The dehydrated samples were embedded in paraffin and subsequently cut into 7 µm sections. H&E staining was performed using a standard protocol with Hematoxylin and Eosin for morphology analysis.

Mitochondrial potential measurement
Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM) was used to measure the mitochondrial potential. PV-ADSCs were treated as indicated and at the day of assay, staining medium (αMEM with 10% FBS and 100 mmol/L TMRM) was added to the cells. After incubation for 30 mins, the cells were detached with trypsin and subjected to fluorescent detection with BD LSR Fortessa flow cytometer.

Trans-well Assay
Migration assays were performed using transwell inserts with 8.0 µm pore membrane filters (Corning,3422). Cultured PV-ADSCs (10 5 cells/ml serum-free medium) were seeded into the upper chamber of the trans-well. The bottom chamber contained serum-free medium with control or murine SDF-1 (Peprotech) at different concentrations. Serum free medium served as negative control. After 16-h incubation, non-migrating cells on the upper side of the filters were carefully washed and removed using a swab. The migrated cells on the lower surface of transwell filter were fixed in 4% PFA for 10 min and then stained with 1% crystal violet (Sigma, HT90132) for 15 min. Images were acquired using Nikon Eclipse TS100 microscope. Cells were counted in 5 random fields under the microscope for statistical analysis.

Cytokine profile of PV-ADSCs
Various cytokines in cell lysates of SC-ADSCs and PV-ADSCs were detected with Proteome Profiler Mouse Angiogenesis Array Kit (R&D systems, ARY015) following the manual.

Dpp4
Icam1   A, Cultured PV-ADSCs on the 2 nd day of cell seeding were subjected to H 2 O 2 treatment for two days (n=3). Data are presented as mean±SD. B, Cultured PV-ADSCs on the 2 nd day of cell seeding were transfected with 12.5 nmol/L Nox4 siRNA for two days (n=2). Data are presented as mean±SD. C, Treatment of cultured PV-ADSCs with (concentration) FCCP for 2 days with or without TGFb1 did not show potent effect in altering SMC marker (Cnn1, Tagln and Acta2) expression (n=3). DMSO, dimethyl sulfoxide; FCCP, carbonyl cyanide-4-phenylhydrazone. Data are presented as mean±SD. *P<0.05. D, Phosphoenolpyruvate and Lactate level after treatment of ADSCs for 4 days with 5 ng/ml TGFb1 (n=3). Data are presented as mean±SD. *P<0.05. E, The ECAR rate at the basal level and after the injection of oligomycin, FCCP, anti-mycin A and rotenone was measured in PV-ADSCs cultured with or without TGFb1 for 1 day. ECAR, extracellular acidification rate; Oligo, oligomycin; Anti-A/Rot, anti-mycin A/Rotenone; FCCP, carbonyl cyanide-4-phenylhydrazone. F, Glucose uptake was measured in PV-ADSCs treated with 5 ng/ml TGFb1 for 2 days and did not show much change in comparison with cells without TGFb1 treatment (n=3). G, Treatment of 25 mmol/L glucose for two days did not induce significant change of SMC markers at mRNA level in PV-ADSCs (n=3).  Figure 7F (n=3). B, Statistic analysis of Figure 7I (n=1). C, The level of miR-378a-3p was detected with Taqman miRNA assay 2 days after TGFb1 treatment (n=3). Data are mean±SD. *P<0.05 and ***P<0.001.  Tables  Supplemental Table I