Animals and ethics statement
The Animal Care and Utilization Committee of the Shanghai Tenth People’s Hospital approved all animal procedures. We obtained male Sprague-Dawley female rats at eight weeks of age from the SLAC Laboratory Animal Co. Ltd, Shanghai, China, and individually housed them in independent ventilated cages under 24 °C to 26 °C under constant humidity with a 12-hour light/dark cycle. All procedures were approved by the Ethics Committee of the the Shanghai Tenth People's Hospital, Shanghai, and were conducted by following the guidelines. We conducted surgical processes under anesthesia and made every effort to minimize suffering. We anesthetized rats by intraperitoneal injection of 30 mg/kg sodium pentobarbital before sacrifice and the rats were sacrifice by spinal dislocation.
ADSC isolation, culture, and identification
Briefly, we harvested adipose tissue from normal rats or healthy humans. The Ethics Committee of the Shanghai Tenth People's Hospital, Shanghai approved all procedures (Human20170621 for humans, Animal20170513 for animals). We conducted procedures by following the guidelines. We conducted surgical processes under anesthesia and made every effort to minimize suffering. We washed tissues with phosphate-buffered saline (PBS) and minced them before digestion with 0.2% collagenase I (Sigma-Aldrich, St. Louis, USA) for 1 hour at 37 °C with intermittent shaking. We washed the digested tissue with Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, USA) containing 15% FBS (Gibco BRL, Frederick, USA) and centrifuged the samples at 1000 rpm for 10 minutes to remove mature adipocytes. We resuspended the pellets in DMEM with 15% FBS, with penicillin (100 U/ml) and streptomycin (100 μg/ml), and cultured cells at 37 °C and 5% CO2. We detached ADSCs reaching 80%–90% confluence with 0.02% ethylenediaminetetraacetic acid/0.25% trypsin (Sigma-Aldrich, St. Louis, USA) for 5 minutes at room temperature and replated them. We utilized fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies against CD29, CD44, CD90, CD105, and von Willebrand factor (vWF) for phenotypic analysis. We used IgG-matched isotype as the internal control for each antibody. We grew normoxic ADSC cultures in 95% air (20% O2) and 5% CO2.
Multilineage differentiation of ADSCs
To validate ADSC multilineage differentiation, we cultured third-passage mouse ADSCs in adipogenic differentiation medium (Sigma-Aldrich, St. Louis, USA) and stained them with oil red O after 2 weeks, or cultured them in osteogenic differentiation medium (Sigma-Aldrich, St. Louis, USA) and stained them with alizarin red after 3 weeks.
ADSC-derived exosome isolation and identification
After reaching 80%–90% confluence, ADSCs were rinsed with PBS and cultured in FBS-free endothelial cell growth medium-2MV, supplemented with 1× serum replacement solution for another 2 days. We removed conditioned culture medium and centrifuged the cells at 300 g for 10 minutes and at another 2000 g for 10 minutes to remove cellular debris and apoptotic cells. After centrifugation at 10,000 g for 0.5 hour, we filtered the supernatant (0.22 μm, Millipore, Billerica, USA), transferred 15 mL of supernatant to an Amicon Ultra-15 Centrifugal Filter Unit (100 kDa, Millipore) and centrifuged it at 4000 g to concentrate the sample to ~1 ml. We washed the ultrafiltration unit twice with PBS and filtered the samples again at 4000 g to achieve a 1 ml volume. We added a 20% volume of Exoquick exosome precipitation solution (System Biosciences, USA) to the ultrafiltered liquid and mixed the sample through inversion. After incubation for half a day, we centrifuged the mixture at 1500 g for 0.5 hour and aspirated the supernatant. We resuspended the exosome pellets in 500 μL PBS. We performed all procedures at 4°C. We determined the exosome protein content with a Pierce bicinchoninic acid Protein Assay Kit (Thermo Fisher Scientific, USA). We stored exosomes at −80 °C until use. We utilized western blotting and transmission electron microscopy to analyze the exosomes. We determined size by dynamic light scattering with a Nanosizer. The size distribution was plotted with the particle radius (nm) on the X-axis and the percentage on the Y-axis.
The miRNA target of SIRT1 were predictions with the targetscan (http://www.targetscan.org/).
We cultured HSFs and HEK293T (FuHeng Biology, Shanghai, China) in high glucose DMEM (Gibco BRL, Grand Island, USA) with 10% FBS. We cultured human mammary endothelial cells (HMECs, Cell Bank of the Chinese Academy of Sciences, Shanghai, China) in MCDB131 medium (Gibco BRL) containing 10% FBS, 2 mM L-glutamine (Sigma-Aldrich), epidermal growth factor (Sigma-Aldrich, 10 ng/ml) and hydrocortisone (1 μg/ml, Sigma-Aldrich). We maintained cells under 37 °C and 5% CO2 in a humidified environment. HMECs and HSFs were stimulated with 5.5 mM or 30 mM glucose for 1 day, and 100 μg/ml of exosomes was added to the cultures to assess protection against high glucose injury.
Exosome labeling and uptake
We used a PKH67 fluorescent linker kit (Sigma-Aldrich, St. Louis, USA) to label exosomes. We added PKH67 dye (400 μL) to exosome suspensions and incubated the samples for 5 minutes at room temperature. We added an identical volume of exosome-depleted bovine serum albumin to stop the reaction, and washed exosomes twice with PBS to remove any unbound dye. We incubated HSFs or HMECs with exosomes labeled for 3 hours, fixed and stained samples with diamidinophenylindole (DAPI), and observed and photographed them under a confocal microscope.
RNA interference or overexpression
We purchased miR-138 inhibitors from RiboBio (Guangzhou, China), and conducted transfection according to standard procedures. In brief, we transferred cells to culture plates with six wells and transfected them through incubation in complete medium containing ADSC-exosomes (200 μg/well, at 100 μg/ml) and Lipofectamine 2000 (Thermo Scientific), or an equivalent volume of PBS for 1 day. For miR-138 overexpression, ADSCs were transfected with an miR-138 mimic (5′-AGCGUGUGUUGUGAAUCAGGCCG-3′) synthesized by GenePharma (Shanghai, China) by Lipofectamine 2000, as described previously.
Quantitative real-time polymerase chain reaction (qPCR)
We extracted RNAs from cells with a TRIzol reagent kit (Invitrogen, Carlsbad, USA), by following the standard procedures. We amplified cDNAs and performed reverse transcription with a TaqMan miRNA reverse transcription kit. The primers used to assay SIRT1 (Gene ID: 309757) expression were reverse, 5′-CACTTTCATCTTCCAAGGGTTC-3′ and forward, 5′-TATTCCACGGTGCTGAGGTA-3′. The PTEN (Gene ID: 50557) primers were reverse, 5′-AAGAGTCAAACAATGGCAAGC-3′ and forward, 5′-TAGTTGGAGTCACCAGGATG-3′. The miR-138 (accession number: MIMAT0000844) primers were reverse, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCGGCCTGATTC-3′ and forward, 5′-ACACTCCAGCTGGGAGCTGGTGTTGTGTTG-3′. qPCR was conducted with a TaqMan human miRNA assay kit. We used 2−ΔΔCT method to capture relative fold differences in expression. U6 and GAPDH were used as internal references.
We used flow cytometry to assay HMECs apoptosis after FITC-conjugated annexin V and propidium iodide (PI) staining. We washed cells twice before adjusting them to 1×106 cells per ml in cold D-Hanks buffer. We added annexin V–FITC (10 μl) and PI (10 μl) to 100 μL cell suspension and incubated them for 15 minutes at room temperature in the dark. Before analysis, we added 400 µl binding buffer to each sample without washing. We performed each assay in at least triplicate.
Tubule formation assay
In vitro neovascularization was assayed in matrices of human fibrin. After treatment, we seeded serum-starved HMECs in endothelial basal medium onto plates coated with Matrigel (105 cells per well into six well plates) (BD Biosciences, Franklin Lakes, USA) and incubated them at 37°C for 12 hours. We observed tubular structures that formed in the Matrigel and photographed under phase-contrast microscopy, and the newly formed tube lengths in ten randomly selected fields per well were measured.
We treated cells with exosomes, miR-138–exosomes, or miR-138 inhibitor under HG conditions. After 24 h, we starved the cells in serum-free medium for another 12 hours, performed trypsin digestion, and seeded 1×105 cells in the top chambers of 24 well Transwell culture inserts (Promega, Fitchburg, WI, USA). Medium with 20% serum was used as a chemoattractant. We fixed the cells for 10 min with formalin of 4% after 24 h culture.
Cell counting kit (CCK)-8 assay
We assayed HSF and HMEC proliferation with a CCK-8 kit (BD Biosciences, Franklin Lakes, USA) by using standard procedures. We cultured transfected cells in 96-well plates with exosomes under HG conditions for 24 h before adding 90 μL fresh culture medium and 10 μl CCK-8 reagent. We detected the absorbance at 450 nm with a microplate reader after incubation at 37°C for 2 hours.
Luciferase reporter assay
The 3′-UTR target sequence for miR-138 miRNA in the SIRT1 gene was predicted with the argetScan online tool. Wild-type and 3′-UTR mutant SIRT1 were performed and cloned into the pMIR firefly luciferase-expressing vector. We cotransfected HEK293T cells at 70% confluence with 500 ng of pMIR-SIRT1-wt/pMIR-SIRT1-Mut and 50 nM of miR-138 mimics with a Lipofectamine 2000 transfection kit (Thermo Scientific) for the luciferase assays. We assayed luciferase activity with the Dual-Luciferase Reporter System (Promega). We independently performed five assays.
Rat diabetes skin wound model
We induced diabetes in rats through a single intraperitoneal injection of 100 mg/kg in 0.01 M pH 4.3 sodium citrate. We measured the blood glucose daily and controlled it to between 16.7 and 33.3 mmol/l through administration of insulin at 6–18 U/day (Wan-Bang Biochemical Medicine Co. Ltd, Xuzhou, China). After 1 month, we established the subcutaneous wound model after isoflurane inhalation anesthesia (2%). We made a single round full-thickness skin wound on the dorsal hind foot with a disposable 5 mm skin biopsy punch and Westcott scissors. Eighteen rats were allocated randomly to subcutaneously injection of 200 μg ADSC-exosomes in PBS of 100 μl or the same volume of PBS at four sites around the wound (25 μl per site). The rats were killed after 15 days, and skin specimens were harvested for histopathological evaluation. Each group have 6 rats. We anesthetized rats by intraperitoneal injection of 30 mg/kg sodium pentobarbital before sacrifice and the rats were sacrifice by spinal dislocation.
Measurement of wound contraction
On days 0, 7, and 15 before wound harvest, the sizes of the treated and control wounds were measured with a ruler and photographed (DMC-LX5GK, Panasonic, Japan). We calculated the ulcer area with Image-Pro Plus 4.5 (http://www.mediacy.com/imageproplus).
Immunohistochemistry and immunofluorescenceassays
We fixed skin tissue samples in 10% formalin solution, embedded them in paraffin, and sectioned them at 5 μm. We stained tissue sections with Masson’s trichrome for histological evaluation. We performed immunofluorescence staining of CD31 to measure histopathological alterations in angiogenesis and used terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to detect apoptotic cells. We examined sections with a fluorescence microscope (Nikon, Tokyo, Japan) or an Axiophot light microscope (Zeiss, Oberkochen, Germany) and photographed them with a digital camera.
Western blot assay
We lysed tissues or cells, and centrifuged lysates at 12,000 rpm under 4°C after adding a protease inhibitor. We determined protein concentrations via a Pierce bicinchoninic acid assay kit (Thermo Fisher). We separated proteins by 10% SDS-PAGE and transferred them to PVDF membranes. To assay protein expression, we used primary antibodies against CD63 (1:600), CD9 (1:600), CD31 (1:1000), CD81 (1:600), SIRT1 (1:500), CD34 (1:1000), PTEN (1:500), p-Akt (1:500), Bax (1:200), caspase-3 (1:600), p-ERK1/2 (1:500), Akt (1:500), ERK1/2 (1:500, all Santa Cruz Biotechnology, Dallas, USA), and GAPDH (1:1000, Sigma-Aldrich). We also used horseradish peroxidase-conjugated secondary antibody (1:1000, Abcam, USA). We used an ECL chemiluminescent kit (Millipore, Burlington, MA, USA) to detect bands.
We denoted continuous variables as means ± SD (standard deviation). We performed one-way analysis of variance for comparisons in GraphPad Prism (GraphPad, La Jolla, USA). A p-value ≤ 0.05 was considered to indicate a significant difference.