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M. Rudnicki et al. / European Journal of Pharmacology 782 (2016) 98–106
2.3. Cell culture
intracellular diacetates, after which 5 M of either GQ-32, GQ-169,
μ
LYSO-7, or RSG was added to the wells. Fluorescence (emission
wavelength, 485 nm; excitation wavelength, 535 nm) was mea-
sured temporally at 37 °C from 0 to 4 h using the bottom-reading
mode in a Synergy MX plate reader (BioTek, Winooski, VT, USA).
The NO concentration in the culture medium was evaluated by
Human umbilical vein endothelial cells (HUVECs) were main-
tained in RPMI 1640 medium, and human embryonic kidney 293
cells (HEK293) were cultured in high-glucose Dulbecco's modified
Eagle's medium (DMEM). Media were supplemented with 10%
ꢀ
fetal bovine serum (FBS), 2 mM
L-glutamine, 100
μ
g/ml strepto-
measuring the NO2 accumulation with a nitric oxide analyzer
mycin, and 100 IU/ml penicillin. Cells were incubated at 37 °C with
5% CO2 and stimulated with GQ-32, GQ-169, and LYSO-7 in med-
ium containing 1% FBS. Control cells were treated with vehicle,
DMSO, at concentrations used for the TZDs (o0.1% vol/vol).
(NOA™ 280; Sievers Inc., Boulder, CO, USA) as described pre-
viously (Bonini et al., 2002). In brief, HUVECs were grown to
confluence in six-well plates and then incubated with or without
5
μ
M GQ-32, GQ-169, LYSO-7, or RSG for 24 h, and 100 l of cul-
μ
ture supernatants were injected into a reflux chamber containing
vanadium(III) in 3 N HCl heated to 90 °C. The NO produced was
detected by gas phase chemiluminescence after reaction with
ozone. A calibration curve then was created using a sodium nitrate
standard solution.
2.4. Cell viability assays
To assess the new TZDs effects on cell viability, a 3-(4,5-di-
methylthiazol-2yl)-2,5-diphenol-2H-tetrazolium bromide (MTT)
reduction assay was conducted (Denizot and Lang, 1986). In brief,
HUVECs were seeded in a 96-well tissue culture plates (5 ꢁ 103
2.8. Migration assay
cells/well) and treated with 0.625, 1.25, 2.5, 5, or 10 M GQ-32,
μ
GQ-169, LYSO-7, or RSG for 24 h. The cells then were incubated
with MTT (1 mg/ml) at 37 °C for 3 h and lysed in 100 l DMSO. The
absorbance was evaluated at 570 nm using a Synergy MX plate
reader (BioTek, Winooski, VT, USA). To study the effects of new
Endothelial cell migration was analyzed using an in vitro
scratch assay as described previously (Liang et al., 2007). In brief,
in vitro scratched wounds were created by scraping the endothelial
confluent monolayers with a sterile tip. After injury of the
monolayer, the cells were washed gently and incubated for 24 h
μ
TZDs on cell death, HUVECs were treated with 5
μ
M GQ-32, GQ-
169, LYSO-7, or RSG for 24 h, and samples were analyzed by flow
cytometry with Canto II (Becton Dickinson, Franklin Lakes, NJ,
USA) using annexin V-FITC apoptosis detection kit (Sigma-Aldrich,
St. Louis, MO, USA), according to the manufacturer's instructions.
with RPMI 1640, 1% FBS containing 5 M GQ-32, GQ-169, LYSO-7,
μ
or RSG. Immediately after wounding and at the end of the ex-
periment, the wound areas were photographed. The AxioVision
4.0 software (Carl Zeiss Inc., Jena, Germany) was used to determine
the extent of migration by determining the uncovered area in
three distinct microscopic fields, representative of each culture
condition. Each experiment was performed in triplicate and re-
peated three times.
2.5. Transient transfection and reporter gene assay
The pCMX expression plasmids for PPAR
γ
and the minimal
promoters containing multiple binding sites for the PPAR-response
element (UAS-LUC) were kindly provided by Dr. A. Castrillo (In-
stituto de Investigaciones Biomédicas Alberto Sols). In brief,
HEK293 cells seeded in 48-well plates (5 ꢁ 104 cells/well) were
transiently transfected using Lipofectamine 2000 reagent (Life
Technologies, Carlsbad, CA, USA) according to the manufacturer's
instructions. Eight hours after transfection, cells were treated with
vehicle, GQ-32, GQ-169, LYSO-7, or RSG and further incubated for
16 h. Reporter activities were assayed using the dual luciferase/
renilla assay system following the recommendations of the sup-
plier (Promega, Madison, WI, USA). Green fluorescent protein was
used as a control for transfection efficiency.
2.9. Matrigel assay
HUVECs (1 ꢁ 105/well) were plated on Matrigel (BD Bios-
ciences, Mountain View, CA) and allowed to adhere for 2 h at
37 °C. GQ-32, GQ-169, LYSO-7, or RSG then was added at 5 M.
μ
Digital photographs of the cells were collected using a Nikon
Eclipse TS100 microscope (Nikon Ltd., London, United Kingdom).
Each experiment was performed in triplicate, and five random
pictures were taken of each well at a magnification of ꢁ 10. The
tube formation was quantified using U.S. National Institutes of
Health ImageJ program (Carpentier, 2012). Mean values of total
length in each sample were used to represent the tube formation.
2.6. Measurement of intracellular ROS
2.10. Assessment of anti-inflammatory activity
To determine the effect of new TZDs on oxidative stress, the
intracellular formation of ROS was detected using the ROS-sensi-
tive fluorescent probe CM-H2DCFDA as described previously (Fu-
jisawa et al., 2009). Cells were incubated in RPMI 1640 medium
For the evaluation of the new TZDs effects on the adhesion
molecules expression, HUVECs were incubated with new TZDs
(5
μ
M) for 4 h in the absence or presence of TNF- (10 ng/ml). The
α
with 5.6 or 30 mM glucose plus 5
μM GQ-32, GQ-169, LYSO-7, or
gene expression of VCAM-1, ICAM, and E-selectin mRNAs were
analyzed by real-time polymerase chain reaction (PCR).
RSG. The cells then were washed, and intracellular ROS formation
was assayed by CM-H2DCFDA oxidation-based fluorescence.
Fluorescence was measured in a Synergy MX plate reader (BioTek,
Winooski, VT, USA).
2.11. Quantitative real-time PCR analysis
Total RNA was isolated with Trizol (Life Technologies, Carlsbad,
2.7. Measurement of intracellular NO production and release
CA, USA), and 2 g RNA was reverse transcribed into cDNA by
μ
SuperScript VILO cDNA synthesis kit (Life Technologies). Quanti-
tative real-time PCR was performed in an ABI Prism 7500 Fast
Sequence Detector (Applied Biosystems, Foster City, CA, USA)
using SYBR Green PCR Master Mix (Applied Biosystems) and spe-
cific primer pairs listed in Table 1. The cycling program was 95 °C
for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s
Each sample was run in triplicate. Expression of each target gene
was normalized to 18S rRNA relative expression as an internal
Intracellular NO measurement was performed as described
previously using the NO-specific fluorescence probe DAF-FM dia-
cetate. In brief, HUVECs were incubated in RPMI 1640 medium
containing 5 M DAF-FM diacetate for 45 min at 37 °C in the dark.
μ
After loading, cells were rinsed three times with PBS to remove
excess probe and then incubated for an additional 30 min with
fresh medium to allow complete de-esterification of the
Rudnicki, Martina
