Reagents and antibodies, RNA isolation and quantitative real-time PCR for miRNAs or P27KIP1 (also known as CDKN1B) and P57KIP2 (also known as CDKN1C) expression, western blot analysis and immunofluorescence analysis are described in detail in the electronic supplementary material (ESM).
Isolation and culture of ECs
ECs were isolated from human umbilical vein within 4 h of delivery by trypsin treatment (0.1% [wt/vol.]), cultured in M199 with the addition of 20% (vol./vol.) bovine calf serum (BCS) and 5 ng/ml of basic fibroblast growth factor (bFGF) and used at early passage (II–III). Throughout the study ECs were cultured for 2 days in normal medium (5 mmol/l d-glucose) plus 10% (vol./vol.) BCS and bFGF (5 ng/ml) alone or in combination with 400 μg/ml AGE, 25 mmol/l d-glucose or 19 mmol/l d-mannitol (used as osmotic control). In selected experiments, ECs exposed to normal medium, AGE or high glucose were transfected for 48 h with pre-miRNA-negative control, pre-MIR221 or pre-MIR222 precursor oligonucleotides or, alternatively, with anti-miRNA-negative control, anti-MIR221 or anti-MIR222 antagonists (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions.
Isolation, characterisation and culture of EPCs from peripheral blood mononuclear cells
The following method was used to isolate EPCs. Peripheral blood mononuclear cells (PBMNCs) were obtained by Ficoll Histopaque 1077 (Sigma-Aldrich, St Louis, MO, USA) and plated onto collagen-1-coated dishes for 21 days in EGM-2 medium (Cambrex, Walkersville, MD, USA), as described by Yoder et al. . FACS analysis was used to characterise EPC surface markers (anti-CD45, anti-CD14, anti-CD34, anti-CD31, anti-kinase insert domain receptor (KDR) and anti-CD146) at day 0 (2 days after isolation), when non-adherent cells were removed and at day 23, at the end of the experiments (21 days of EGM-2 culture plus 2 days with the stimuli: 400 μg/ml AGE, 25 mmol/l d-glucose glucose (‘high glucose’) or 19 mmol/l d-mannitol (‘high mannitol’)). The control conditions were 5 mmol/l d-glucose (‘normal’). Approval was obtained both from Servizio Immunoematologia e Medicina Trasfusionale and from the Institutional Review Board of S. Giovanni Battista Hospital, Turin, Italy. Informed consent was provided according to the Declaration of Helsinki. In selected experiments, EPCs exposed to normal medium, AGE or high glucose were transfected for 48 h with pre-miRNA-negative control, pre-MIR221 or pre-MIR222 precursor oligonucleotides or, alternatively, with anti-miRNA-negative control, anti-MIR221 or anti-MIR222 antagonists (Applied Biosystems), according to the manufacturer’s instructions.
To analyse cell-cycle progression, ECs and EPCs treated for 2 days as indicated were processed by FACS analysis, as previously described by Defilippi et al. . Briefly, after treatment, the cells were fixed with 70% (vol./vol.) ethanol and DNA was stained with propidium iodide (Sigma-Aldrich) and analysed with a flow cytometer (FACScan, Becton Dickinson, San Jose, CA, USA). The percentage of cells in each phase of the cell cycle was determined by ModFit LT software (Verity Software House, Topsham, ME, USA). The percentage of the cells in the DNA duplicating phase (S phase) was reported. Cell-cycle analysis by FACS was also performed on: (1) ECs treated with different AGE concentration (from 50 to 1,200 μg/ml); and (2) ECs and EPCs transfected for 48 h with pre-miRNA-negative control, pre-MIR221 or pre-MIR222 precursor oligonucleotides or, alternatively, with anti-miRNA-negative control, anti-MIR221 or anti-MIR222 antagonists in normal conditions or in the presence of AGE, high glucose or high mannitol.
In vitro endothelial cell migration assay
Analysis of chemotaxis of ECs was performed as previously described by Brizzi et al. and Dejana et al. [23, 24]. Briefly, assessment of EC migration was performed in Boyden’s chambers by counting the cells that passed across the filter (8 μm pore size) after addition in the lower compartment of the chamber of the vehicle alone (free medium with 0.25% [wt/vol.] BSA), high glucose (25 mmol/l), high mannitol (19 mmol/l), or AGE (400 μg/ml), in the presence of VEGF (20 ng/ml). Cell counting was performed by three different operators on 10 fields, ×20 magnification, of three individual experiments (n = 9).
Luciferase miRNA target reporter assay
The luciferase reporter assay was performed using a construct generated by subcloning the PCR products amplified from full-length 3′-untranslated region (UTR) of P27KIP1 and 3′-UTR of P57KIP2 DNA in the SacI restriction site of the luciferase reporter vector plasmid miR (pmiR) (Ambion, Applied Biosystems). The PCR products were obtained using the following primers: P27KIP1 sense 5′-AGAGCTCCAGATACATCACTGC-3′, antisense 5′-TGAGCTCTATACTTGGCTCAG-3′; P57KIP2 sense 5′-TTGAGCTCCCCTTCTTCTCGCTGTCCTCT-3′, antisense 5′-AAGAGCTCCTCTTTGGGCTCTAAATTGGC-3′.
The insert identities were verified by sequencing. The pmiR, pmiR-3′-UTR-P27KIP1 and pmiR-3′-UTR-P57KIP2 reporter vectors were transiently co-transfected in ECs and EPCs, cultured in normal medium alone or in combination with 400 μg/ml AGE or 25 mmol/l d-glucose or 19 mmol/l d-mannitol, at 30:1 molar ratio with the pRL vector, coding for the Renilla sp. luciferase, used as the internal control of the luciferase assay. Luciferase activities were analysed 48 h later by Dual-Luciferase Report Assay System (Promega, Madison, WI, USA), according to the vendor’s instructions, using a TD20/20 double injector luminometer (Turner Designs, Forlì, Italy). The results were expressed as relative luciferase activity (%), calculated by normalising the ratio of the firefly/Renilla sp. luminescences. Luciferase activities, using the pmiR reporter vectors, described above, were also evaluated in ECs or in EPCs, transfected 24 h previously with pre-miRNA negative control, pre-MIR221 or pre-MIR222 precursor oligonucleotides.
In vivo experiments
For the murine angiogenesis assay FVB mice (five mice, 8 weeks old, for each experimental group) (Charles River Laboratories International, Wilmington, MA, USA) were injected s. c. with Matrigel containing VEGF (50 ng/ml) [25, 26], high glucose (25 mmol/l), and AGE (400 μg/ml) alone or in combination as indicated. The negative control was NaCl solution (154 mmol/l). After 7 days, the FVB mice were killed and Matrigel plugs were processed for histological analysis with haematoxylin–eosin staining. In selected experiments for the angiogenesis assay, SCID mice (five mice, 7 weeks old, for each experimental group) (Charles River Laboratories) were injected s.c. with growth-factor-reduced Matrigel containing VEGF, AGE and 2 × 106 ECs, previously transfected with pre-miRNA negative control, pre-MIR221 or pre-MIR222 precursors and processed as described by Zeoli et al. .
Briefly, 4 days after injection Matrigel plugs were recovered and fixed in 10% (vol./vol.) buffered formalin and embedded in paraffin for histological and immunofluorescence analyses or digested for EC isolation. The vessel area and the total Matrigel area were planimetrically assessed from haematoxylin–eosin-stained sections as previously described by Zeoli et al. . Only the structures possessing a patent lumen and containing erythrocytes were considered vessels. Angiogenesis was expressed as the percentage ± SD of the vessel area relative to the total Matrigel area (% vessel area, ×10 magnification). Quantification of neo-formed vessels was also evaluated by CD31 staining of vascular ECs. Any stained EC or EC cluster, clearly separated from connective tissue elements, was considered as a single microvessel and counted according to Weidner et al. . Animal procedures conformed to the Guide for Care and Use of Laboratory Resources (National Institutes of Health publication no. 93–23, revised 1985) .
Isolation of ECs from Matrigel plugs
ECs were recovered from Matrigel plugs 4 days after injection into SCID mice . After digestion in Hank’s buffered salt solution containing 0.1% (vol./vol.) collagenase I for 30 min at 37°C, the cells were washed in medium plus 10% (vol./vol.) BCS and forced through a graded series of meshes to separate the cell component from Matrigel matrix. ECs were isolated via anti-human CD31 antibody coupled to magnetic beads, by magnetic cell sorting using the MACS system (Miltenyi Biotec, Auburn, CA, USA). Briefly, cells were labelled with the anti-human CD31 antibody for 20 min and then were washed twice and re-suspended in MACS buffer (PBS without Ca2+ and Mg2+, supplemented with 1% [vol./vol.] BSA and 5 mmol/l EDTA) at the concentration of 0.5 × 106 cells/80 μl. After washing, cells were separated on a magnetic stainless steel Wool column (Miltenyi Biotec) according to the manufacturer’s recommendation. The endothelial phenotype was verified by FACS analysis using an anti-human von Willebrand antibody (Sigma-Aldrich). The recovered cells were subjected to RNA isolation to detect MIR221 and MIR222 expression by quantitative real-time PCR or lysed for western blot analysis.
All in vitro and in vivo results are representative of at least three independent experiments, performed in triplicate. Densitometric analysis using a BioRad GS 250 molecular imager was used to calculate the differences in the fold induction of protein content, reported as ‘densitometric value’ and each western blot panel and relative densitometric histogram shown in the figures was representative of the results obtained in triplicate. The significance of differences between experimental and control values for both in vitro and in vivo experiments was calculated using analysis of variance with Newman–Keuls multi-comparison test and reported in detail in each figure legend (n = 9).