Animals
Four-week-old wild-type (WT) C57/BL mice and eight-week-old T2DM mice C57BL/KsJ-db/db (db/db) were purchased from Shanghai Research Center for Model Organisms (Shanghai, China) and reared specific pathogen-free. All the in vivo animal experiments were approved by the Animal Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.
Isolation, culture, and characterization of ADSCs
The subcutaneous adipose tissue was acquired sterile from the areas of groin and armpits of 4-week-old male WT C57/BL6 mice, digested using NB4 collagenase (Nordmark, Uetersen, Germany), shaken to form a homogenous mixture, and seeded on culture plates with Dulbecco’s modified Eagle medium (DMEM) containing normal glucose (5.5 mmol/L), 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin for primary culture. The cultures were incubated at 37°C under 5% CO2. The ADSCs of the third passage were collected, washed with phosphate-buffered saline (PBS), and incubated with anti-mouse antibodies of CD29, CD90, CD45, and CD34 for 25 minutes at 4°C in the dark. The isotype antibodies were used as negative controls. Flow cytometry (Beckman Coulter, Fullerton, CA, USA) was performed on the cells after washing 3 times with PBS as described previously [21].
Glyoxalase-1 overexpression in the ADSCs
To stably obtain large numbers of ADSC-derived exosomes, lentiviral vectors were used for gene transfection of ADSCs. The lentiviral vectors containing GLO-1 and GFP (green fluorescent protein) gene were purchased from Hanbio Biotechnology (Shanghai, China). The lentivirus was incubated with the ADSCs for 24 hours at a multiplicity of infection (MOI) of 30. The expression of GFP in the ADSCs was observed using a fluorescence microscope (Olympus IX81, Tokyo, Japan, http://www.olympus-ims.com) to identify the transfection efficiency. The expression of the GLO-1 in the G-ADSCs was confirmed at the levels of protein and mRNA by Western Blot and qPCR, respectively.
Isolation and characterization of the G-ADSC-Exos
After the successful production of the ADSCs stably overexpressing the GLO-1, the G-ADSC-derived exosomes (G-ADSC-Exos) were isolated as previously described [22]. First, the cell samples were centrifuged at 300 x g for 10 minutes and the supernatant was collected. Then the garnered supernatant was centrifuged at 2,000 x g for 10 minutes and the supernatant was collected. Finally, the garnered supernatant was centrifuged at 10,000 x g for 3 hours and the supernatant was discarded while saving the pellet. After resuspension of the precipitate with PBS, nanoparticle tracking analysis (NTA) (ZetaView, Particle Metrix, Meerbusch, Germany) was performed to measure the diameter of the exosomes. The morphological characteristics of the exosomes were observed using a transmission electron microscope (TEM) (JEM-2100F, Japan Electronics, Tokyo, Japan). The exosomal markers such as CD9, CD63, CD81, and TSG101 and the expression of the GLO-1 in the G-ADSC-Exos were looked at using the Western blot.
Co-culture of HUVECs and G-ADSC-Exos under high glucose environment
Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Cell Resource Center at the Institute of Life Sciences (Shanghai, China), and cultured with high glucose (33.3 mmol/l) DMEM with 5% fetal bovine serum (Gibco, Waltham, MA, USA) and 1% antibiotic/antimycotic solution (Gibco) as previously described [23]. The HUVECs were then co-cultured with CM-Dil labeled G-ADSC-Exos at 37°C under 5% CO2 for 24 h. The G-ADSC-Exos engulfed by the HUVECs were observed using a fluorescence microscope (Olympus IX81, Tokyo, Japan, http://www.olympus-ims.com). The DAPI was used to stain nuclei, and phalloidin was used to stain the cytoskeletons of the HUVECs.
Cell proliferation and apoptosis assay of HUVECs
The cell counting kit-8 (CCK-8; Abcam, Cambridge, UK, https://www.abcam.cn) was used to identify the most effective therapeutic concentration of the G-ADSC-Exos on the HUVECs. The HUVECs were seeded on 96-well plates at a density of 2×103 cells/well in high glucose conditions and cocultured with PBS, 25 µg/mL ADSC-Exos, 50 µg/mL ADSC-Exos, 100 µg/mL ADSC-Exos, 25 µg/mL G-ADSC-Exos, 50 µg/mL G-ADSC-Exos and 100 µg/mL G-ADSC-Exos, respectively. The CCK-8 solution was added to the wells at 10 µL/well at 0, 24, 48, and 72 h, after the incubation for 2 h at 37°C. A microplate spectrophotometer (Varioskan; Thermo Fisher Scientific, Eugene, OR, USA) was employed to measure the optical density (OD) at 450 nm wavelength.
For apoptosis assay, the HUVECs were cocultured for 48 h with PBS, 100 µg/mL ADSC-Exos and 100 µg/mL G-ADSC-Exos under high glucose conditions, respectively. The Annexin V PE/7-AAD apoptosis detection kit (Solarbio Science & Technology Co. Ltd., Beijing, China) and flow cytometry (Beckman Coulter, Fullerton, CA, USA) were used to measure the apoptosis ratio as previously mentioned [24]. Finally, the Western blot assay was performed on the lysate of the HUVECs cocultured with PBS, ADSC-Exos, and G-ADSC-Exos using antibodies against Caspase-3, Bcl-2, Bax, and β - actin (1:500; Abcam, Cambridge, UK).
Wound healing, transwell migration, and tube formation assay of the HUVECs
To elucidate how the G-ADSC-Exos influenced the migration and angiogenic ability of the HUVECs under high glucose conditions during the process of wound healing, transwell migration and tube formation assay were performed as previously described [21]. For the wound-healing assay, after scraping and washing with PBS, the HUVECs and 100 µg/mL G-ADSC-Exos were co-incubated under a high glucose environment for 24 h, and then the co-culture was viewed using an inverted microscope (Olympus, Tokyo, Japan, http://www.olympus-ims.com) at 0 and 24 h. The Image J software (National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/) was used to measure the area of the gaps.
The transwell migration assay was performed with a Boyden chamber and a polyethylene terephthalate (PET) membrane (R&D Systems Inc., Minneapolis, MN, USA). 100 µg/mL G-ADSC-Exos and 2×103 HUVECs were added onto the upper chamber containing 100 µL high glucose DMEM with 0.5% FBS, and a 500 µL high glucose DMEM was added to the lower chamber. After 24h, a cotton swab was used to wipe the Matrigel and cells from the upper chamber, 4% paraformaldehyde was used to fix the HUVECs migrated through the PET membrane, and then 1% crystal violet in 2% ethanol was used to stain the fixed HUVECs. An inverted microscope (Olympus, Tokyo, Japan, http://www.olympus-ims.com) was adopted to capture the images.
For the tube formation assay, the HUVECs were cocultured with 100 µg/mL G-ADSC-Exos in high glucose DMEM for 48 h, then seeded on Matrigel in a culture dish and incubated at 37°C for 12 h. An inverted microscope (Olympus, Tokyo, Japanhttp://www.olympus-ims.com) was adopted to capture the images, and the ImageJ software (National Institutes of Health, Bethesda, USA, https://imagej.nih.gov) was utilized to calculate the cumulative tubular growth. All the experiments mentioned above were performed three times.
Construction of T2DM mouse limb ischemia model and treatment of limb ischemia
Twenty-four male 8-week-old T2DM C57BL/KsJ-db/db mice (Shanghai Research Center for Model Organism, China) were anesthetized with the intraperitoneal injection of 0.3 ml/kg of 1% chloral hydrate. The skin of the left hindlimb was shaved, disinfected with iodophor, and the sterile drapes were placed. A longitudinal incision of 5 mm from the groin to the inner thigh was made, the membranous vascular sheath was gently pierced to expose and separate the femoral artery, vein, and nerve under a 20-fold Olympus SZ61 stereoscopic microscope (Olympus, Tokyo, Japan, http://www.olympus-ims.com). The femoral artery was ligated at the distal end of the common femoral artery and the proximal end of the superficial femoral artery with 7 − 0 surgical sutures, respectively. This model by a simple low ligation of the femoral artery was reported to mostly mimic clinical peripheral vascular disease, and suitable for studying the regeneration of blood vessels and striated muscle in the field of regenerative medicine [25]. The 24 mice were randomly divided: the ADSC-Exo group (n = 8), the G-ADSC-Exo group (n = 8), and the PBS control group (n = 8). After 24 h, the sites of the gastrocnemius, gracilis, and quadriceps muscles in the three groups received injections of 2 mL PBS with 100 µg/mL ADSC-Exos, 2 mL PBS with 100 µg/mL G-ADSC-Exos and 2 mL PBS, respectively. The blood flow was evaluated with a laser Doppler perfusion imager (moorFLPI; Moor Instruments, Devon, UK) noninvasively on the first, seventh, and 28th day after implantation.
Histological analysis
The mice from the three groups were anesthetized and perfusion-fixed on day 28. The hindlimb muscles were harvested and the Masson’s trichrome staining was applied to evaluate the structural integrity of the ischemic muscles. The immunohistochemical and immunofluorescence staining were adopted to observe the density of microvessels as described previously [21]. For the immunofluorescence staining of the muscle sections, the α-smooth muscle actin (α-SMA) was stained with FITC (Abcam), and the nucleus was stained with DAPI (Dako).
Detection of paracrine factors in the G-ADSC-Exos
The protein expression of tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) in the ADSC-Exos and G-ADSC-Exos were measured using ELISA kits (R&D Systems Inc., Minneapolis, MN, USA) in vitro. All the experiments mentioned above were performed three times.
Detection of ROS production in the HUVECs
After cocultured under high glucose conditions with PBS, 100 µg/mL ADSC-Exos and 100 µg/mL G-ADSC-Exos for 48 h, respectively, the HUVECs were labeled with 2, 7-dichlorofluorescein diacetic acid (DCFH-DA), and the level of intracellular ROS (reactive oxygen species) was detected with a ROS analysis kit (Beyotime, Shanghai, China). Briefly, the adherent HUVECs were incubated with the DCFH-DA at a final concentration of 5 mM at 37°C for 20 min and then washed three times with PBS. An inverted fluorescence microscope (Olympus IX81, Tokyo, Japan, http://www.olympus-ims.com) was used to analyze the production level of ROS in the HUVECs immediately.
Detection of signaling pathways in vitro and in vivo
For the in vitro experiment, total protein was isolated from the HUVECs cocultured with PBS, 100 µg/mL ADSC-Exos and 100 µg/mL G-ADSC-Exos under high glucose conditions for 48 h, respectively. And for in vivo experiment, total protein was isolated from the muscle tissues of the ADSC-Exo group, the G-ADSC-Exo group, and the PBS group, respectively, and the proteins were probed with the following antibodies: anti-eNOS and p-eNOS antibodies, anti-AKT and p-AKT antibodies, anti-ERK antibody, anti-P-38 and p-P-38 antibodies, and anti-AP-1, anti-ASC, anti-Caspase-1, anti-NLRP3, anti-IL-1-β and anti-β-actin antibodies (1:500; Abcam). Signals were detected after incubating with an HRP-labeled secondary antibody and a chemiluminescent substance (Roche, Basel, Switzerland), and then the images were collected with a LAS3000 machine (GE Healthcare Life Sciences, Pittsburgh, USA).
Statistical analysis
The mean b± standard deviation was used to describe parametric values. The one-way analysis of variance and two-tailed Student’s t-test were performed to compare data between groups using GraphPad Prism version 6.0 (GraphPad, La Jolla, CA, USA, http://www.graphpad.com) and SPSS version 25.0 (IBM-SPSS Inc., Armonk, NY, USA, https://www.ibm.com). A p < 0.05 was defined as statistically significant.