INTRODUCCIÓN: REVISIÓN CRITICA DEL PROBLEMA

ANEXOS
Medical Association Declaration of Helsinki. Briefly, umbilical veins were digested
with collagenase IA (22 U mL -1 , Gibco, Invitrogen) in a 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) buffer solution (HBS) of the following
composition (in mmol · L -1 ): 110 NaCl, 5 KCl, 0.5 NaH2PO4, 0.5 KH2PO4, 0.16
CaCl2, 2 MgCl2, 10 NaHCO3, 0.5 EDTA, 10 HEPES, 10 glucose (pH 7.4). After 6
min digestion at 37 ºC, endothelial cells were collected, washed and placed in a
chemically defined endothelial cell medium (PAA, Pasching, Austria)
supplemented with 10 ng · mL -1 of β–endothelial cell growth factor (Sigma-Aldrich),
10 μg · mL -1 of heparin (Mayne Pharma, Spain) and with 100 U · mL -1 penicillin
(Laboratios Normon, Madrid, Spain) and 100 μg · mL -1 streptomycin (Laboratorio
Reig Jofré, Barcelona, Spain). Finally, HUVEC were cultured on 0.2% (w/v) gelatin
(Sigma-Aldrich) pre-coated flasks or dishes (Falcon, BD Biosciences) and grown
to confluence in a humidity saturated 5% CO2 atmosphere at 37 ºC. Confluence
cultures were detached with 0.25% (w/v) trypsin in 1 mmol · L -1 of EDTA in Hanks'
balanced salt solution (Gibco) and cells for the experiments were used between
fourth to tenth passage.
For the experiments, HUVEC were placed at a concentration of 10,000 cells or
300,000 per well in 96-wells or 6-wells microplates, respectively, with
supplemented endothelial cell growth medium. 10,000 HUVEC per well (96 wells
plate) were used for extracelular ROS measurement and 300,000 HUVEC per well
(6 wells plate) were employed for RNA isolation and protein extraction. Previously
to the experiments, we made a 12h serum and supplements starvation period.
Then, quiescent HUVEC were treated with gHSA or human serum albumin (HSA)
at 25 μg · mL -1 for 4 hours. All incubations were carried out at 37 ºC in 5%
humidified CO2 environment. None of the treatments affected cellular viability of
HUVEC, as determined by cell count, morphology and trypan blue exclusion.
b) RNA isolation and polymerase chain reactions (PCR)
After the treatments cell culture medium was aspirated and floating cells were
removed. Total cellular RNA from HUVEC was isolated with NucleoSpin®
(Macherey-Nagel) according to the manufacturer’s protocol and quantified with a
spectrophotometer (Nanodrop ND-1000, Thermo Fischer). One μg of the isolated
total RNA was reverse transcribed in a total volume of 30 μL, using random
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