A total of 40 subjects were included in the current study, including 25 patients with PAD who were diagnosed via the ankle-brachial index or angiography. Patients with acute coronary syndrome, acute ischemic limb or malignancy were excluded. A total of 15 healthy individuals without any evidence of PAD served as the control group. Blood sampling was performed and samples were subjected to RNA isolation (FocusGenomics Biotech Co., Ltd, Taiwan). Following miRNA array analysis, we selected specific miRNAs for comparison between the patients with PAD and the control group. This study was approved by the Taipei Veterans General Hospital’s Institutional Review Board for Research (approval no. 2013-08-020B#3), and written informed consent was provided by all the participants prior to their inclusion within the study.
MiRNA library construction and next-generation sequencing
RNA was isolated according to the manufacturer’s protocol. Total RNA from samples was further constructed into libraries using an Illumina TruSeq Small RNA Library Prep kit (Cat. RS-200-0012; Illumina, San Diego, CA, USA). In brief, 1 μg of high-quality total RNA from each sample was ligated with adapters using T4 RNA ligase. The adapter-ligated RNA samples were reverse-transcribed to cDNA, amplified by primers containing a specific sequence index, and then size-validated using an Agilent 2100 bioanalyzer (Cat. G2943, Agilent, Santa Clara, CA, USA) loaded with a DNA 1000 kit (Agilent, cat. 5067-1504). The size-checked libraries were loaded onto Novex TBE gels (Cat. EC6265BOX, Thermo Fisher Scientific, Inc., Waltham, MA, USA), and then size-selected and gel-eluted to obtain proper fragments. The eluted libraries were qualified using an Agilent 2100 bioanalyzer loaded with a DNA 1000 kit, and quantified using Qubit (Thermo Fisher Scientific, Inc., Cat. Q33216) and real-time PCR (Q-PCR). Each library was diluted in equal concentrations and the same volumes were taken for pooling. Pooled libraries were sequenced for 10 M reads/samples with a high-throughput, 50-bp single-end sequencing reagent on an Illumina MiSeq sequencing system.
Scratch injury model and EPCs
Human EPC isolation, cultivation and characterization were performed as previously described . EPC migration was evaluated using a scratch injury model. EPCs were transfected with miR-548j-5p mimic or antagomir, or a control. After serum-starvation of EPCs overnight, a scratch injury was applied with a scalpel, and EPC sprouting was examined before and 12/24 h after scratching.
Tube formation assay
An EPC tube formation assay was performed using an in vitro angiogenesis assay kit (Chemicon, Temecula, CA, USA). EPCs transfected with miR-548j-5p mimic or antagomir were placed onto a matrix with medium for 16 h. Tubule formation was inspected by inverted light microscopy. Six random fields were used to calculate the average number of complete tubes formed by cells using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).
Western blot analysis
EPCs were lysed in a lysis buffer (62.5 mM Tris-HCl, 2% sodium dodecyl sulfate, 10% glycerol, 0.5 mM phenylmethanesulfonyl fluoride, 2 μg/mL aprotinin, pepstatin, and leupeptin). Proteins in the cell lysates were separated using sodium dodecyl sulfate-polyacrylamide (10%) gel electrophoresis, followed by transfer onto poly (vinylidene fluoride) membranes. The membranes were then probed with monoclonal antibodies against phosphorylated eNOS (Upstate Biotechnology, Lake Placid, NY, USA), SDF-1 and actin. Protein band densitometry was performed using ImageQuant software (Promega, Madison, WI, USA).
Directed in vivo angiogenesis assay (DIVAA)
Angiogenesis in vivo was evaluated using a DIVAA kit (Trevigen, Gaithersburg, MD, USA). Briefly, angioreactors were filled with EPCs transfected with 50,000 miR-548j-5p mimic, negative control, miR-548j-5p antagomir or scramble control and VEGF/FGF1 embedded in 20 μl basement membrane extract .
Angioreactors were incubated at 37°C for 1 h. For positive controls, angioreactors were filled with BME supplemented with VEGF (12.5 ng/ml) plus FGF1 (37.5 ng/ml). Two angioreactors were implanted in each immunocompromised nude mouse (eight- week-old male nude mice) subcutaneously in the dorsal region. The angioreactors were removed 14 days after implantation and photographed. The presence of blood vessels was assessed using FITC-Lectin detection, and fluorescence was determined using a plate reader in mean relative fluorescence units.
Ischemic hind-limb model and EPC transplantation
Eight-week-old male nude mice were purchased from the BioLASCO Taiwan Co., Ltd. After 2 weeks observation, unilateral hind-limb ischemia was induced by left femoral artery ligation. EPCs transfected with miR-548j-5p mimic (n=6) or antagomir (n=6) or a control (n=6) were injected intramuscularly at six different sites on the ischemic limb distal to the arterial occlusion site. The blood flow of the hind limb was measured using a Laser Doppler perfusion imaging system (Moor Instruments Limited, Devon, UK) before and after surgery, and then weekly. The results were expressed as the ratio of perfusion in the ischemic vs. the non-ischemic limb.
The mice were sacrificed 3 weeks after surgery and the limbs were fixed overnight in methanol. The ischemic muscles were embedded in paraffin, and then deparaffinized to incubate them with rat monoclonal antibodies against murine CD31 (BD PharMingen, San Diego, CA, USA). New capillaries were identified based on morphology and positive staining for CD31 using the avidin-biotin-complex technique and Vector Red Chromogenic substrate (Vector Laboratories, Burlingame, CA, USA) after counterstaining with hematoxylin. The visible capillaries were counted under 10 random fields, and the capillary density was expressed as the number of capillaries/mm.
All animals were housed and handled in accordance with the criteria outlined in the National Institutes of Health ‘‘Guide for Care and Use of Laboratory Animals”. The study protocol was approved by the Institutional Animal Care and Use Committee of Taipei Veterans General Hospital, Taipei, Taiwan (approval number: IACUC_2013–076).
Data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using the unpaired Student’s t-test or one-way analysis of variance, followed by Scheffe’s multiple comparison post hoc test using Statistical Package of the Social Sciences software (version 14; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.