Study design and participants
The observational study was conducted in Guangzhou, China from 2020.02 to 2020.10. Sixty-three outpatients diagnosed as new onset of T2DM were enrolled into DM group and 60 matched (age, education, economy, diet, etc.) healthy subjects were recruited into control group. The inclusion criteria for T2DM were as follows: male, aged from 18 to 70, diagnosed with type 2 diabetes for less than 3 months according to American Diabetes Association diagnostic glucose criteria [19]. Patients with a history of T1DM, malignancy, hyperlipidemia, liver disease, chronic renal failure, autoimmune diseases, or active inflammatory disease and those with other comorbidities were excluded. Finally, as the flow chart shown (Fig. S1), fifteen patients from T2DM group receive dapagliflozin (10 mg once daily) for 3 months to control blood sugar according to the desire of patients and the International Diabetes Federation Guidelines for treatment [20]. The study was approved by the ethics committee of our hospital and registered at URL: https://www.clinicaltrials.gov; Unique identifier: ChiCTR2100047733.
Optical coherence tomography angiography (OCTA)
OCTA was performed using OptovueAngioVue system (RTVue XR Avanti, Optovue, Inc., Fremont, U.S). In brief, the operator started scanning by using a light source centered on 840 nm and bandwidth of 50 nm in order to obtain a high-quality OCT images on the monitor. Retina capillary density (RCD) and retinal non-perfused areas (R-NPA) measurements were performed on 3 mm×3 mm scan field size and were used to assess ocular perfusion impairment in vascular conditions. RCD was a measurement of the proportion of pixels occupied by flowing vessels out of the total pixels within the area of analysis. R-NPA was automatically calculated by the software, while the outputs were adjusted by mouse click selection. The reliability of the intravital capillaroscopy and optical coherence tomography angiography has been confirmed by our previous studies [18, 21].
EPCs culture, identification, and materials
Circulating EPCs were quantitated with CD34+ KDR+ or CD34+ CD133+ cells per 100 thousand peripheral blood mononuclear cells (PBMCs) by flow cytometry analysis (Beckman Coulter, Fullerton, CA, USA) in accordance with our early reports [22]. EPCs were isolated and cultured as previously described [23]. After 14 days of culture, EPCs were defined as cells double positive for 1,1′-dioctadecyl-3,3,3,3′ tetramethylindo-carbocyanine (DiI) acetylated low density lipoprotein (ac-LDL) uptake (Invitrogen, Carlsbad, CA, USA) and fluorescein isothiocyanate (FITC) lectin binding (Sigma-Aldrich, St. Louis, MO, USA). EPCs were pre-treated with/without dapagliflozin (50 µM) for 24 h in the presence/absence of AMPK activator AICAR (AI) or inhibitor compound C (cC), (100 µM).
EPCs migration assay
EPCs migration was determined using a modified Boyden chamber. In brief, 2×104 EPCs,resuspended in 250 µL endothelial basal medium (EBM-2) (Lonza Clonetics, San Diego, CA, USA), were pipetted in the upper chamber (Costar Transwell assay, 8 µm pore size; Corning, NY, USA). The chamber was placed in a 24-well culture dish containing 500 µL EBM-2 supplemented with either PBS, or 100 ng/mL stromal cell-derived factor 1 (SDF1, Peprotech, Rocky Hill, NJ, USA). After 24 h incubation at 37℃, transmigrated cells were counted by independent investigators blinded to treatment randomly.
EPCs adhesion to endothelial cells assay
A monolayer of Human umbilical vein endothelial cells (HUVECs) was prepared 48 h prior the assay by plating 2×105 cells in each well of a four-well plate. HUVECs were pretreated with or without 1 ng/mL fibronectin (Peprotech Inc, Rocky Hill, NJ, USA) for 12 h. Then, 1×105 CM-DiI (CellTracker™ CM-DiI; Invitrogen)-labeled EPCs were added to each well and incubated for 3 h at 37℃. Nonattached cells were gently removed by PBS, and adherent EPCs were fixed with 4% paraformaldehyde and counted by independent investigators.
EPCs vascular tube formation assay
EPCs (2×105 cells/well) were seeded onto Matrigel (400 µL per well; BD Pharmingen, Bedford, MA) in a 4-well chamber slide and coated overnight in EBM-2 containing VEGF (20 ng/mL; Lonza Clonetics, San Diego, CA, USA) to induce tube formation. After the incubation at 37°C with 5% CO2 for 12 h, the EPCs formed capillary-like vascular network on the Matrigel. EPCs were stained with fluorescent calcein AM (Molecular Probes, Carlsbad, CA, USA) and visualized by a confocal microscope (Olympus, Tokyo, Japan). Tube lengths were measured using the imaging analysis software (ImageJ; National Institutes of Health). The averages in three low-power fields randomly selected were calculated per experiment. All assays were performed on at least five independent cultures.
Mouse hindlimb ischemia model and in vivo vasculogenesis assay
Male NRMInu/nu athymic nude mice (SLAC laboratory animal center, Shanghai, China) aged 8 to 10 weeks. All experimental protocols complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH) and the Animal Care and Use Committees of Sun Yat-Sen University. Unilateral hindlimb ischemia surgery was induced in male NRMInu/nu athymic nude mice by resecting the right femoral and saphenous arteries following the intraperitoneal administration of an anesthetic agent (50 mg/kg of pentobarbital sodium). To assess the vasculogenic capacity of EPCs, Cultured EPCs (1×105 cells of each) were intramuscularly injected into three sites of ischemic gastrocnemius muscle. DMSO (0.1%) treated cells were used as controls. Hindlimb blood perfusion was measured using a laser Doppler perfusion imaging system (Moor Instruments, UK) weekly. The results were presented as the blood flow ratio of the right (ischemic) to left (non-ischemic) limb. Three weeks after surgery, the mice were euthanized by intraperitoneal injection of an overdose of pentobarbital, and their hindlimb muscles were harvested for further analyses.
Histochemical staining and capillaries density analysis
Samples from the ischemic hindlimb were harvested and embedded in paraffin after fixation in 4% formaldehyde. Sections (5 µm) were deparaffinized and stained with hematoxylin & eosin (HE) to estimate the degeneration and damage of fibers by morphology. Three different fields from each muscle were randomly selected. Ischemic damage was semi-quantitatively. (0 = no difference from the normal, 1 = mild discoloration, 2 = moderate discoloration, 3 = severe discoloration or subcutaneous tissue loss or necrosis, and 4 = any amputation). For capillaries density assay, muscle samples were collected and digested for 1 h at 37°C in an enzymatic mix (10 mg/ml Collagenase type II (C6885, Sigma Aldrich) and 1 mg/mL Elastase (LS002292, Worthington Biochemistry) to single cell and incubated with rat anti-mouse CD31 antibody (BV421, BD Pharmingen) for 20 min followed by incubation with biotinylated secondary antibody. Flow cytometry analysis was performed for the percentage of CD31 positive cells.
RT-PCR and western blot analysis
Total RNA was extracted with the mRNA purification kit (Takara Biotecnology, Dalian CO., LTD.) according to the manufacture’s protocol. RT-PCR was carried out by the routine 2-step method. EPC proteins were harvested by cell lysis buffer (Cell Signaling Technology, Beverly, USA). Protein extracts were subjected to SDS-PAGE, transferred to polyvinylidene fluoride membranes (Roche, Indianapolis, USA). The following antibodies were used: rabbit anti- AMPK antibodies (1:1000; abCAM, Cambridge, UK), rabbit anti-phospho-AMPK antibody (1:1000; abCAM), rabbit anti-actin antibody (1:2000; Cell Signaling Technology). Proteins were visualized with HRP-conjugated anti-rabbit IgG (1:3000; Cell Signaling Technology), followed by use of the ECL chemiluminescence system (Cell Signaling Technology).
Determination of AMP/ATP ratios
Adenine nucleotides from perchloric acid extracts of EPCs were analyzed by ion-exchange chromatography. The areas under the AMP and ATP peaks were integrated and used to calculate the AMP/ATP ratio. In each case the values are the mean ± SD from three to four independent measurements.
Measurement of intracellular Ca2+
Intracellular Ca2+ levels were determined with the Ca2+ sensitive fluorochrome Fluo-3/AM. EPCs were incubated with 3 uM Fluo-3/AM at 37°C for 30 min in the dark after the treatment with salidroside plus H2O2. Cells were then gently rinsed three times and the fluorescence was analyzed by spectrophotometer (PerkinEl mer LS 55).
Statistical analysis
Continuous variables and category variables are presented as mean ± SD or n (%), respectively. A Student’s t test or ANOVA was performed to compare the difference of baseline characteristics and the change of parameters before and after intervention. A value of two-sided P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 22.0 software (IBM Corporation, Somers, New York, USA) and figures were plotted using Graphpad Prism software (version 9.0).