MiR-146a as marker of senescence-associated pro-inflammatory status in cells involved in
vascular remodelling
Fabiola Olivieri1,2, Raffaella Lazzarini1,2, Rina Recchioni2, Fiorella Marcheselli2, Maria Rita
Rippo1, Silvia Di Nuzzo1, Maria Cristina Albertini3, Laura Graciotti1, Lucia Babini1, Serena
Mariotti2, Giorgio Spada4, Angela Marie Abbatecola5, Roberto Antonicelli6, Claudio
Franceschi7 and Antonio Domenico Procopio1,2.
1-Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy
2-Center of Clinical Pathology and Innovative Therapy, IRCCS-INRCA, National Institute, Ancona, Italy
3- Dipartimento di Scienze Biomolecolari, Sezione di Biochimica e Biologia molecolare, Università degli Studi di
Urbino ‘‘Carlo Bo’’, Urbino, Italy Cardiology Unit, I.N.R.C.A. National Institute, Ancona, Italy
4- Dipartimento di Scienze di Base e Fondamenti, Università degli Studi di Urbino ‘‘Carlo Bo’’, Urbino, Italy
5- Scientific Direction, IRCCS-INRCA, National Institute, Ancona, Italy
6- Cardiology Unit, IRCCS-INRCA, National Institute, Ancona, Italy
7-Department of Experimental Pathology, “Alma Mater Studiorum” University of Bologna and Centro
Interdipartimentale Galvani “CIG”, Alma Mater Studiorum University of Bologna, Bologna, Italy
Corresponding author:
Fabiola Olivieri, Ph.D,
Dept. Clinical and Molecular Science, Università Politecnica delle Marche, Ancona
Via Tronto 10/A - 60020 Ancona (Italy)
Ph: +39-071-2206242, Fx: +39-071-2206240, email: f.olivieri@univpm.it
Authors mailing list: Raffaella Lazzarini = raffaellalazzarini@hotmail.com; Rina Recchioni =
r.recchioni@inrca.it; Fiorella Marcheselli = f.marcheselli@inrca.it; Maria Rita Rippo =
m.r.rippo@univpm.it; Silvia Di Nuzzo = silviadinuzzo@libero.it; Maria Cristina Albertini =
maria.albertini@uniurb.it ; Laura Graciotti = l.graciotti@univpm.it; Lucia Babini =
l.babini@univpm.it; Serena Mariotti = s.mariotti@inrca.it; Angela Marie Abbatecola=
angela_abbatecola@libero.it; Roberto Antonicelli = r.antonicelli@inrca.it; Claudio Franceschi =
claudio.franceschi@unibo.it; Antonio Domenico Procopio = a.d.procopio@univpm.it.
Material end Method
Cell Culture
HUVECs (Human Umbilical Vein Endothelial Cells), HAECs (Human Aortic Endothelial Cells)
and HCAECs (Human Coronary Artery Endothelial Cells) were purchased from Clonetics
Corporation (Lonza) and cultured in endothelial basal medium EBM, supplemented with EGM-2 or
EGM-2/MV Single Quots containing 0.1% rh-EGF (human recombinant epidermal growth factor),
0.04% hydrocortisone, 0.1% VEGF (vascular endothelial growth factor), 0.4% rh-FGF-B (human
recombinant fibroblast growth factor), 0.1% rh-IGF-1 (insulin- like growth factor) 0.1% ascorbic
acid, 0.1% heparin, 0.1% GA-1000 (gentamicin sulfate plus amphotericin B) and 2% or 5% fetal
calf serum.
Fresh cells were plated at a seeding density of 2.500 cells/cm2 in T 75 flasks and the medium was
changed every 48 hours. Cell cultures reached confluence after 6-7 days, as assessed by light
microscopic examination, and they were passaged at weekly intervals. After trypsinization and
before replating, harvested cells were counted using a haemocytometer and the number of
population doublings (PD) were calculated using the following formula: (log10F – log10I) x 3,32
(where F indicates the number of cells at the end of the passage and I the number of cells when
seeded) (Maciag et al., 1981).
Endothelial cell aging was studied by subjecting endothelial cells to subsequent passages until
passage XIII, as previously described (Haendeler et al., 2004). Cumulative population doubling
(CPD) was calculated as the sum of all the changes in PD.
Senescence Associated β-Galactosidase (SA-β-Gal) Staining
Non confluent HUVEC cells cultured on 6 well plate were washed twice in phosphate-buffered
saline (PBS), pH 7,4 and fixed for 10 minutes in 2% formaldehyde and 0,2% glutaraldehyde in
PBS. After two washes in PBS, cell cultures were incubated for 18 hours at 37°C with freshly
prepared β- Gal staining solution containing 1mg/ml 5-bromo-4-chloro-3indolyl- β-D-
galactopyranoside (X-gal.), 5mM potassium ferro cyanide, 5mM potassium ferro cyanide, 150mM
NaCl, 2mM MgCl2, 40mM citric acid, titrated with NaH2PO4
to pH 6.0. β- Gal was
microscopically revealed by the presence of a blue, insoluble precipitate within the cell, the
percentage of SA- β-gal positive cells was determined by counting at least 500 cells in each sample
(Dimiri et al., 1995).
Telomere length determination
Telomere length analysis was performed by using both the traditional absolute quantification
method, based on Southern analysis of terminal restriction fragment (TRF) (A), and relative
telomere length analysis, based on quantitative PCR Cawthon’s method (B). (Cawthon et al., 2002).
Genomic DNA was isolated from HUVECs using a DNA Isolation Kit (Qiagen, Valencia, CA).
A) Absolute telomere length determination in HUVECs, HCAECs and HAECs, was assessed by the
terminal restriction fragment length assay using a TeloTAGGG Telomere Length Assay Kit (Roche
Applied Science, Indianapolis, IN).
In brief DNA was extracted from cells and digested by an optimized mixture of frequently cutting
restriction enzymes, which digest non-telomeric-DNA fragments. The DNA fragments were
separated by gel electrophoresis and transferred to a nylon membrane by Southern blotting. The
blotted DNA fragments were hybridized to a digoxigenin (DIG)-labeled probe specific for telomeric
repeats and incubated with a DIG-specific antibody covalently coupled to alkaline phosphate, which
in turn, metabolizes CDP-Star, a highly sensitive chemiluminescent substrate. The visible signal
produced indicates the location of the immobilized telomere probe (and, hence, the TRF). The
average TRF length was determined by comparing the location of the TRF on the blot relative to a
molecular weight standard.
B) Relative telomere length determination, applied to CHF and CTR CACs, was performed as
previously described (Testa et al., 2011; Olivieri at al., 2011).
Telomerase (TERT) activity assay
Quantitative determination of telomerase activity was performed by using a quantitative telomerase
detection (QTD) kit (Allied Biotech, Inc., Ijamsville, MD), a validated, high-sensitive real time
quantitative telomeric repeat amplification protocol (RQ-TRAP), according to the manufacturer’s
protocol (Wege et al., 2003).
In brief, cells were lysed in 1X lysis buffer and incubated on ice for 30 min. The lysate was then
centrifuged at 12,000 g for 30 minutes at 4°C, and the supernatant collected. The protein
concentration of the cell lysate was determined with Bradford’s assay method. All amplifications
were performed on a iQ5 real-time PCR detection system (Bio-Rad Laboratories, Inc.) Telomerase
activity was expressed relatively to a standard curve obtained from serial dilution of telomere short
repeat (TSR) control template. Standards, inactivated samples and no-template-reactions were also
assayed on every plate for quality control purposes. Melting curve analysis was performed on each
run to verify specificity and identity of the PCR products.