Z. Benslimane-Ahmim et al.
Mechanistic study of the proangiogenic effect of osteprotegerin
Title: Mechanistic study of the proangiogenic effect of osteoprotegerin
Zahia BENSLIMANE-AHMIM, Florence POIRIER, Claudine DELOMENIE, Anna LOKAJCZYK,
Françoise GRELAC, Isabelle GALY-FAUROUX, Amel MOUHAMEDI, Anne-Marie FISCHER,
Dominique HEYMANN7, Didier LUTOMSKI, and Catherine BOISSON-VIDAL.
Supplemental methods for online publication
Real-time quantitative PCR (RT-qPCR)
Total RNA was isolated from cells using the RNAble reagent (Eurobio, Les Ulis, France), according
to the manufacturer's instructions. RNA concentration was measured by reading the absorbance at 260
nm using a BioMate3 spectrophotometer (Thermo Fisher Scientific), and purity was checked by
measuring absorbance at 230 and 280 nm. RNA integrity evaluated by capillary electrophoresis using
the 2100 Bioanalyzer and RNA 6000 Nano chips (Agilent Technologies) was considered suitable
when RIN>8.0 (RNA Integrity Number). For quantification of mRNA expression, single-stranded
cDNA was reverse-transcribed from 1 µg of total RNA, with random hexamers and oligo-dT priming
using the iSCRIPT enzyme (Bio-Rad), according to the manufacturer's instructions. A 50-fold dilution
of each cDNA was amplified in duplicate in a CFX96 real-time thermal cycler (Bio-Rad), using the
SsoFast EvaGreen Supermix reagent (Bio-Rad) according to manufacturer’s instructions, and 500 nM
final concentration of each primer. The amplification specificity was checked by melting curve
analysis and agarose gel electrophoresis of PCR products. Standard curves were established for each
gene from serial dilutions of cDNA from control cells, to check that PCR efficiency was at least 90%.
GeNorm algorithm in qBase PLUS tool was used to select GAPDH and RPLP0 genes as references for
normalization of mRNA expression results. The transcripts of reference genes were quantified in each
cDNA sample to account for sample-to-sample differences in RNA concentration, quality, or reverse
transcription efficiency. The relative amount of RNA expression in samples was determined using the
2-
Cq
method where
Cq = (Cqtarget – Cqreference)sample - (Cqtarget – Cqreference)calibrator, the calibrator
being the control (untreated) cells condition. Final results were expressed as the n-fold differences in
target gene expression relative to control (untreated) cells, yielding gene expression values in arbitrary
units.
In vitro angiogenesis assays
-Cell adhesion assay in static conditions
Recombinant human fibronectin (10μg/mL) and human type I collagen (10μg/mL), were used to coat
96-well culture dishes, after which OPG pretreated ECFCs (0.4nM for 24h) were incubated at a
density of 4x104 cells/well in adhesion buffer (cation-free HBSS, 10mmol/L HEPES, 1mmol/L CaCl2,
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Z. Benslimane-Ahmim et al.
Mechanistic study of the proangiogenic effect of osteprotegerin
1mmol/L MgCl2, 2mg/mL BSA, pH 7.4). After 20min or 2h, plates were washed twice with PBS, and
the number of adherent cells was determined by measuring acid phosphatase activity (pNPP, Sigma) at
405nM (Fluostar optima;BMG labtech).
-Chemotaxis assay
Chemotaxis was examined in 24-well Boyden microchemotaxis chambers (Costar) with 8µm poresize polyvinylpyrrolidone free polycarbonate Nucleopore filters. ECFCs were pretreated with EBM2,
5%FCS (negative control), EBM2, 5%FCS with 12nM SDF-1 (positive control), 0.4nM OPG(1-401),
OPG(1-194), or supernatants of SDF-1/OPG-pretreated ECFC. When required, AMD3100 (10µg/mL)
was added 30min before OPG, or ECFCs were pretreated for 2h at 37°C with a mixture of 0.5U/ml
heparinase I, 0.1U/ml heparinase III and 0.2U/ml ABC chondroitinases before OPG stimulation. 24h
later, 1.5x104 control and pretreated-cells in suspension in EBM2, 5%FCS were placed in the upper
chamber and left to migrate toward EBM2, 5% FCS (negative control), EBM2, 5% FCS with 12nM
SDF-1 (positive control), 0.4nM OPG (1-401) or 0.4nM OPG(1-194). Migration was allowed to
proceed for 6h at 37°C with 5% CO2. Cells remaining on the upper surface of the filters were
mechanically removed, and the filters were then fixed with 1.1% formaldehyde and stained with
Giemsa. The number of migrated cells was determined by counting under a high-power microscope.
-Wound healing assay
HUVECs were seeded on 0.2% gelatin-coated 6-well plastic culture dishes and pretreated with
AMD3100 (10µg/ml) for 30 min. HUVECs layers were wounded by scraping with a pipette tip, then
incubated for 6h with OPG (0.4nM), SDF-1 (12nM) or supernatants of SDF-1/OPG-pretreated
ECFCs. The extent of cell migration into the wounded area was photographed under the microscope
at 0 and 6 hours. The migration rate was determined by measuring the wound surface area, and the
percentage wound closure was calculated as [(wound Area 0h - wound Area 6h) / wound Area
0h]*100.
-In vitro tube formation assay
ECFCs were pretreated with EBM2, 3% FCS (negative control), EBM2, 3% FCS with 0.7nM VEGF
(positive control), 0.4nM OPG (1-401) or 0.4nM OPG (1-194). When required, ECFCs were
pretreated for 2h at 37°C with a mixture of heparinase I, heparinase III and ABC chondroitinases
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Z. Benslimane-Ahmim et al.
Mechanistic study of the proangiogenic effect of osteprotegerin
before OPG stimulation. 24h later, ECFCs were seeded on Matrigel in growth-factor-depleted basal
medium and cultured for 18h at 37°C with 5% CO2. Cells were then fixed with 11% glutaraldehyde
for 15min and stained with Giemsa. The total length of tube structures was quantified over the entire
surface of each well using Videomet software (Microvision Instruments, Paris, France).
Actin polymerisation assay
ECFCs pretreated with OPG (0.4nM for 24h), seeded on multiwell slides, fixed with 4%
paraformaldehyde at room temperature for 15min, then washed with PBS, permeabilized with 0.2%
Triton and stained with FITC-conjugated phalloidin (2mM, Sigman-Beckman Coulter). The cells were
coverslipped with glycerol/PBS containing ToPro3 (invitrogen) in order to identify cell nuclei by
fluorescence microscopy.
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