2.3. hAFS secretome separation and concentration

The total cell secretome, as represented by the cell‐conditioned medium (hAFS‐CM) from either hAFS in control normoxic condition (hAFS‐CM^Normo) or hypoxic preconditioning (hAFS‐CM^Hypo), was collected as previously described.8, 9, 20 Briefly, hAFS‐CM formulations were centrifuged to remove cell debris and further concentrated using ultrafiltration membranes with a 3 kDa selective cut‐off (Amicon Ultra‐15; Millipore) at 4°C first at 3000 ×g for 90’ and then at 3000 ×g for additional 30’. hAFS‐CM protein concentration was assessed by BiCinchoninic Acid (BCA) assay (Gibco—Thermo Fisher Scientific). hAFS‐CM was used for in vitro experiments as 80 mg/mL solution to be added to the cell culture medium as from previous studies.8, 9

Physiological salt solution (PSS) had the following composition (in mmol/L): 150 NaCl, 6 KCl, 1.5 CaCl₂, 1 MgCl₂, 10 Glucose and 10 Hepes. In Ca²⁺‐free solution (0Ca²⁺), Ca²⁺ was substituted with 2 mmol/L NaCl, and 0.5 mmol/L EGTA was added. Solutions were titrated to pH 7.4 with NaOH. The osmolality of PSS as measured with an osmometer (Wescor 5500) was 338 mmol/kg.

Endothelial colony‐forming cells were loaded with 4 µmol/L fura‐2 acetoxymethyl ester (fura‐2/AM; 1 mmol/L stock in dimethyl sulfoxide) in PSS for 1 hour at room temperature. The details of the Ca²⁺ recording set‐up have been described in REF9, 14 and are reported in the Supplementary Information. All the experiments were performed at room temperature. All the data have been collected from ECFCs isolated from peripheral blood of at least three healthy volunteers.

Twenty‐four hours before treatment with hAFS‐CM^Hypo and the specific blockers of intracellular Ca²⁺ signalling, 6 × 10⁴ ECFCs were plated onto 13 mm coverslips in 24‐well plates. ECFCs were fixed in 4% formaldehyde in PBS for 15 minutes at room temperature, permeabilized for 7 minutes in PBS with 0.1% Triton X‐100 and blocked for 30 minutes in 2% gelatin. Then, primary (incubated for 1 hour at 37°C) and secondary (incubated for 1 hour at room temperature) antibodies were applied in PBS with 2% gelatin. The primary anti‐p65 (NF‐κB subunit) antibody specific for immunocytochemistry (Santa Cruz Biotechnology, catalog no. Sc‐372) was used at 1:50 dilution, whereas the AlexaFluor 488 secondary antibody from Invitrogen (catalog no. A‐21441) was used at 1:200. After washing (3 times for 5 minutes each), nuclei were stained with 40,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI) for 15 minutes at room temperature. Fluorescence images were acquired using a Leica epifluorescence microscope equipped with S Fluor X40/1.3 objective using MetaMorph software.

Early passage (P2‐P3) ECFCs were cultured in basal medium EBM‐2 supplemented with 2% FBS in Cultrex (Trevigen)‐coated 96‐well plates, in the absence or in the presence of hAFS‐CM^Hypo for 24 hours. Capillary network formation was assessed starting from 4 up to 24 hours later. The angiogenic response was measured by evaluating both dimensional and topological parameters. The length of endothelial tube‐like structures (tubules or TLS), number of polygon structures established by TLS, referred to as meshes and indicative of endothelial cell migration, and number of master junctions were measured from acquired bright‐field pictures by using the Angiogenesis Analyzer plugin of ImageJ (Gilles Carpentier, Faculte’ des Sciences et Technologie, Universite’ Paris Est, Creteil Val de Marne, France).21, 22 Micrographs were captured by using an Olympus IX71‐inverted microscope (Olympus Europa GmbH) equipped with a CPlan F1 10 ×/0.30 objective. Three different sets of experiments, each performed in duplicate, were carried out. To evaluate the effect of Ca²⁺ signalling, the same protocol was repeated by priming ECFC with hAFS‐CM^Hypo in the presence of RN‐1734 (20 μmol/L), a selective blocker of transient receptor potential vanilloid 4 (TRPV4),22, 23 or thymoquinone (25 μmol/L), a specific NF‐κB inhibitor.12

Fura‐2/AM was obtained from Molecular Probes (Molecular Probes Europe BV). YM‐58483/BTP‐2 (BTP‐2; 4‐methyl‐4'‐[3,5‐bis(trifluoromethyl)‐1H‐pyrazol‐1‐yl]‐1,2,3‐thiadiazole‐5‐carboxanilide) was purchased from Tocris Bioscience. Glycyl‐l‐phenylalanine 2‐naphthylamide (GPN) was obtained from Santa Cruz Biotechnology. All the chemicals were of analytical grade and obtained from Sigma Chemical Co.

All the data have been collected from ECFCs deriving from at least three distinct donors. Pooled data are given as mean ± SE and statistical significance (P < .05) was evaluated by Student's t test or one‐way ANOVA followed by the post hoc Dunnett's test as appropriate. Data relative to Ca²⁺ signals are presented as mean ± SE, while the number of cells analysed is indicated in the corresponding bar histograms.