INTRODUCCIÓN: REVISIÓN CRITICA DEL PROBLEMA

CAPÍTULO IX
e) Measurement of ROS in HUVEC
Extracellular superoxide anion formation was determined by the cytochrome c
reduction method previously described 357 . After treatments, HUVEC were
incubated in a HBS supplemented with 1.5 mmol · L-1 CaCl2 (calcium rich HBS)
and containing cytochrome c (200 μmol · L -1 ). Superoxide production was induced
by adding β- nicotinamide adenine dinucleotide reduced (NADH, 100 μmol · L -1 ).
The specificity for superoxide anion measuring was confirmed by superoxide anion
dismutase (SOD; 100 U · mL -1 ) inhibition, an enzymatic scavenger of these anions.
In a series of experiments cells were preincubated with the following enzyme
inhibitors, BAY11- 7082 2 μmol · L -1 (NF-kB), SP600125 20 μmol · L -1 (AP-1),
LY294002 1.5 μmol · L -1 (PI3K), SL327 10 μmol · L-1 (MEK 1/2) or the inhibitor of
eNOS, N(w)-nitro-L-arginine methylester (L-NAME; 100 μmol · L -1 ) during 30 min
prior to the treatments with HAS or gHSA and maintained during the 4 hours of
these treatments. Cytochrome c reduction was monitored reading the absorbance
at 550 nm on a microplate reader (VersaMax, Molecular Devices) during 60 min at
37 ºC to obtain the ΔAbs550nm/min of each experiment. Amadori products can
autooxidise and reduce the ferrocytochrome c 23 , so we assured that gHSA was not
present in the moment of cytochrome c reduction measurement.
f) Drugs and Materials
The following drugs were purchased from Sigma-Aldrich (Madrid, Spain): HSA,
gHSA (we confirmed that the gHSA was AGE-free measuring the fluorescence at
360/40 Ex - 460/40 Em 17 ), BAY11-7082, cytochrome c, LY294002, NADH, L-
NAME, SL327 and P600125. BAY 11-7082, SP600125, LY 294002 and SL 327
were dissolved in pure dimethyl sulfoxide (DMSO) as stock solutions and then
daily diluted in calcium rich HBS for the experiments. All other reagents were
dissolved directly in calcium rich HBS. Final concentration of DMSO never
exceeded 0.01% (v/v) in the experiments and proper controls were always made
to assess any effect of the vehicle. Collagenase IA was from Gibco (Invitrogen
S.A., Barcelona, Spain). Equipments, mediums and specific reagents or antibodies
employed in the different techniques are indicated in the corresponding sections.
All other reagents used in the experiments and in the preparation of working
solutions were of the best commercially available quality.
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