New thiazolidinediones affect endothelial cell activation and angiogenesis

Article abstract of DOI:10.1016/j.ejphar.2016.04.038

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor-γ (PPARγ) agonists used in treating type 2 diabetes that may exhibit beneficial pleiotropic effects on endothelial cells. In this study, we characterized the effects of three new TZDs [GQ-32 (3-biphenyl-4-ylmethyl-5-(4-nitro-benzylidene)-thiazolidine-2,4-dione), GQ-169 (5-(4-chloro-benzylidene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-dione), and LYSO-7 (5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione)] on endothelial cells. The effects of the new TZDs were evaluated on the production of nitric oxide (NO) and reactive oxygen species (ROS), cell migration, tube formation and the gene expression of adhesion molecules and angiogenic mediators in human umbilical vein endothelial cells (HUVECs). PPARγ activation by new TZDs was addressed with a reporter gene assay. The three new TZDs activated PPARγ and suppressed the tumor necrosis factor α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. GQ-169 and LYSO-7 also inhibited the glucose-induced ROS production. Although NO production assessed with 4-amino-5-methylamino-2′,7′-difluorofluorescein-FM probe indicated that all tested TZDs enhanced intracellular levels of NO, only LYSO-7 treatment significantly increased the release of NO from HUVEC measured by chemiluminescence analysis of culture media. Additionally, GQ-32 and GQ-169 induced endothelial cell migration and tube formation by the up-regulation of angiogenic molecules expression, such as vascular endothelial growth factor A and interleukin 8. GQ-169 also increased the mRNA levels of basic fibroblast growth factor, and GQ-32 enhanced transforming growth factor-β expression. Together, the results of this study reveal that these new TZDs act as partial agonists of PPARγ and modulate endothelial cell activation and endothelial dysfunction besides to stimulate migration and tube formation.

Full text of DOI:10.1016/j.ejphar.2016.04.038

European Journal of Pharmacology 782 (2016) 98–106
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
New thiazolidinediones affect endothelial cell activation and
angiogenesis
Martina Rudnicki a, Gustavo L. Tripodi a, Renila Ferrer a, Lisardo Boscá b, Marina G.R. Pitta c,
Ivan R. Pitta c, Dulcineia S.P. Abdalla a,
n
a Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil
b Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, Spain
c Core of Therapeutic Innovation, Federal University of Pernambuco, Recife, PE, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor-
γ
(PPAR ) agonists used in
γ
Received 25 January 2016
Received in revised form
8 April 2016
Accepted 20 April 2016
Available online 21 April 2016
treating type 2 diabetes that may exhibit beneficial pleiotropic effects on endothelial cells. In this study,
we characterized the effects of three new TZDs [GQ-32 (3-biphenyl-4-ylmethyl-5-(4-nitro-benzylidene)-
thiazolidine-2,4-dione), GQ-169 (5-(4-chloro-benzylidene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-
dione), and LYSO-7 (5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione)]
on endothelial cells. The effects of the new TZDs were evaluated on the production of nitric oxide (NO)
and reactive oxygen species (ROS), cell migration, tube formation and the gene expression of adhesion
Keywords:
Thiazolidinediones
Endothelial cells
Inflammation
Angiogenesis
molecules and angiogenic mediators in human umbilical vein endothelial cells (HUVECs). PPAR
vation by new TZDs was addressed with a reporter gene assay. The three new TZDs activated PPAR
suppressed the tumor necrosis factor -induced expression of vascular cell adhesion molecule 1 and
γ
acti-
and
γ
α
intercellular adhesion molecule 1. GQ-169 and LYSO-7 also inhibited the glucose-induced ROS produc-
tion. Although NO production assessed with 4-amino-5-methylamino-2′,7′-difluorofluorescein-FM probe
indicated that all tested TZDs enhanced intracellular levels of NO, only LYSO-7 treatment significantly
increased the release of NO from HUVEC measured by chemiluminescence analysis of culture media.
Additionally, GQ-32 and GQ-169 induced endothelial cell migration and tube formation by the up-reg-
ulation of angiogenic molecules expression, such as vascular endothelial growth factor A and interleukin
8. GQ-169 also increased the mRNA levels of basic fibroblast growth factor, and GQ-32 enhanced
transforming growth factor-
act as partial agonists of PPAR
besides to stimulate migration and tube formation.
β
expression. Together, the results of this study reveal that these new TZDs
γ
and modulate endothelial cell activation and endothelial dysfunction
& 2016 Elsevier B.V. All rights reserved.
1. Introduction
represent an important target for prevention and treatment of
diabetes-induced vascular complications (Kolluru et al., 2012). In
fact, endothelial dysfunction, which manifests itself by decreased
bioavailability of endothelium-derived nitric oxide (NO), and en-
dothelial cell activation, characterized by increased expression of
cell-surface adhesion molecules, are defining features of vascular
pathologies (Rask-Madsen and King, 2007). Moreover, endothelial
cells are the main cells involved in the formation of new blood
vessels from pre-existing ones, a process termed angiogenesis, and
the abnormalities in this process also contribute to the develop-
ment of vascular complications (Antonetti et al., 2012; Falanga,
2005; Zent and Pozzi, 2007). While deficient angiogenesis is as-
sociated with critical limb ischemia (Falanga, 2005), excessive
angiogenesis is implicated in the pathogenesis of retinopathy
(Antonetti et al., 2012) and nephropathy (Zent and Pozzi, 2007).
The insulin-sensitizing thiazolidinedione (TZD) drugs are ago-
Vascular complications are a leading cause of diabetes-related
morbidity and mortality (Rask-Madsen and King, 2013). Macro-
vascular complications, which mainly result from atherosclerosis,
compromise arteries and augment the chance of critical limb
ischemia, myocardial infarction, and stroke (Laakso and Kuusisto,
2014). In turn, microvascular complications have deleterious ef-
fects on eyes (Antonetti et al., 2012), kidneys (Zent and Pozzi,
2007), and nerves (Boulton et al., 2004) leading to blindness, end-
stage renal disease, and lower leg amputations.
Endothelial cells modulate several vascular functions and
n
Correspondence to: Avenida Professor Lineu Prestes 580, Cidade Universitária,
São Paulo, SP, Brazil.
E-mail addresses: dspa@usp.br, dspabdalla@gmail.com (D.S.P. Abdalla).
nists of the peroxisome proliferator-activated receptor-
γ
(PPAR ),
γ
http://dx.doi.org/10.1016/j.ejphar.2016.04.038
0014-2999/& 2016 Elsevier B.V. All rights reserved.

M. Rudnicki et al. / European Journal of Pharmacology 782 (2016) 98–106
99
a ligand-activated transcription factor of the nuclear hormone
receptor superfamily – and are used clinically for the treatment of
diacetate acetyl ester (CM-H2DCFDA) were obtained from Molecular
Probes Inc. (Eugene, OR, USA). Tissue culture media, serum, and
supplements were purchased from Life Technologies (Carlsbad, CA,
USA) unless otherwise stated.
type 2 diabetes. PPAR
ostasis, and in addition to its role in adipogenesis, strong evidence
supports that PPAR activation also regulates endothelial biology
γ
is a master regulator of energy home-
γ
(Bishop-Bailey and Swales, 2008). Consistently, TZDs affect en-
dothelial cell functions and activation. For instance, TZDs enhance
NO production in endothelial cells (Calnek et al., 2003; Polikan-
driotis et al., 2005), suppress gene expression of adhesion mole-
cules, such as vascular cell adhesion molecule 1 (VCAM-1), inter-
cellular adhesion molecule 1 (ICAM-1) and E-selectin (Kurebayashi
et al., 2005; Pasceri et al., 2000; Wang et al., 2002), and inhibit the
production of reactive oxygen species (ROS) (Ceolotto et al., 2007;
Fujisawa et al., 2009). Furthermore, TZDs also regulate angiogen-
esis in both in vitro and in vivo models (Biscetti et al., 2008; Pa-
nigrahy et al., 2002; Scoditti et al., 2010; Xin et al., 1999). Never-
theless, despite the beneficial effects of TZDs on endothelial cells
homeostasis, these drugs had restricted prescription or have been
withdrawn from the market because of their association with
important adverse effects, including weight gain, liver injury, and
increased risk to bone fracture and heart failure (Cariou et al.,
2012). Such safety concerns have highlighted the need to identify
and characterize new drug candidates from the TZD class of drugs.
In the current study, we sought to investigate the effects of three
new TZDs on endothelial cells, with a special emphasis on the
production of NO and ROS, gene expression of adhesion molecules,
and modulation of cell migration and tube formation and compare
the results to those obtained with rosiglitazone (RSG), which was
used as a TZD control.
2.2. GQ-32, GQ-169, and LYSO-7 synthesis
Thiazolidine-2,4-dione was N-(3)-alkylated in the presence of
sodium hydroxide, which allows the thiazolidine sodium salt to
react with the benzyl halide in a hot ethanol medium, yielding the
first group of intermediates. The 5-arylidene-3-benzyl-thiazoli-
dine-2,4-diones were prepared by a nucleophilic Michael addition
of the 3-benzyl-thiazolidine-2,4-dione and the respective aryl-
substituted ethyl-(2-cyano-3-phenyl)-acrylates to obtain the ar-
ylidene-thiazolidine-2,4-diones 3-substituted. After cooling, the
precipitates were purified by column chromatography or crystal-
lized in suitable solvents. The ethyl-(2-cyano-3-phenyl)-acrylates
were prepared by Knoevenagel condensation of ethyl cyanoacetic
ester and substituted benzaldehydes in the presence of piperidine
(Pitta et al., 2007). All of the synthesized compounds were ana-
lyzed with multiple analytical procedures. Melting points were
determined in a capillary tube using a Quimis apparatus. Infrared
spectra (IR) were recorded on a Bruker IFS66 spectrometer (Bruker
Daltronics, Billerica, MA, USA). 1H NMR spectra were recorded on a
Varian Plus 300 MHz spectrometer using dimethyl sulfoxide
(DMSO)-d6 as a solvent and tetramethylsilane as an internal
standard. Mass spectra were recorded by a high capacity ion trap
mass spectrometer (HCT Ultra; Bruker Daltonics, Billerica, MA,
USA) and were performed by electrospray ionization in negative
mode or by electronic impact. The following results were ob-
tained: GQ-32—C23H16N2O4S. Mp: 152–153 °C. Yield: 52%. IR (KBr,
cmꢀ1): 1738, 1691, 1614, 1410, 1343, 1149, 843. 1H NMR (300 MHz,
2. Materials and methods
2.1. Chemicals and reagents
DMSO-d6)
δ 8.36 (d, 2H, J¼9.0 Hz, 5-Ar-H, 3,5 pos.), 8.09 (s, 1H,¼
CH), 7.91 (d, 2H, J¼9.0 Hz, 5-Ar-H, 2,6 pos.), 7.66–7.62 (m, 4H, 3-Ar-
H, 3,5 pos. and 2′,6′ pos.), 7.48–7.33 (m, 5H, 3-Ar-H, 2,6 pos. and
3′,4′,5′ pos.). MS m/z (%), observed: 167 (100.0), 416 (77.3), 32
(73.8), calculated 416.08; GQ-169–C17H10Cl3NO2S. Mp: 208–210 °C.
Yield: 57%. IR (KBr, cm–1): 1744, 1686, 1611, 1488, 1438, 1378, 1339,
The compounds GQ-32 [3-biphenyl-4-ylmethyl-5-(4-nitro-ben-
zylidene)-thiazolidine-2,4-dione], GQ-169 [5-(4-chloro-benzyli-
dene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-dione], and LYSO-7
[5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazoli-
dine-2,4-dione] (Fig. 1) were synthesized in the Laboratory of Design
and Drug Synthesis of the Federal University of Pernambuco (Recife,
Pernambuco, Brazil). Rosiglitazone (RSG) was purchased from Cay-
1138, 1075, 939, 783, 568, 521. 1H NMR (300 MHz, DMSO-d6)
δ 7.91
(s, 1H,¼CH), 7.62 (d, 4H, J¼2.1 Hz, 5-Ar-H, 2,3,5,6 pos.), 7.50–7.47
(m, 2H, 3-Ar-H, 3,5 pos.), 7.37 (t, 1H, J¼16.2 Hz, J¼7.2 Hz, 3-Ar-H,
4 pos.), 5.09 (s, 2H,-CH2). MS m/z [MꢀH], observed: 397.04, cal-
culated 398.69. The analytical results of LYSO-7 have already been
reported (Santin et al., 2013b).
man Chemicals (Ann Arbor, MI, USA). Tumor necrosis factor
α
(TNF-
α) was obtained from Sigma-Aldrich (St. Louis, MO, USA). 4-Amino-
5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM diace-
tate) and 5-(and-6) chloromethyl-2′,7′ dichlorodihydrofluorescein
Fig. 1. Chemical structures of GQ-32, GQ-169, LYSO-7, and rosiglitazone. GQ-32 [3-biphenyl-4-ylmethyl-5-(4-nitro-benzylidene)-thiazolidine-2,4-dione], GQ-169 [5-(4-
chloro-benzylidene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-dione], and LYSO-7 [5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione] were
synthesized as indicated under Materials and Methods.

100
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

M. Rudnicki et al. / European Journal of Pharmacology 782 (2016) 98–106
101
Table 1
Forward and reverse primers used for real-time PCR.
Gene
Forward primer
Reverse primer
E-selectin 5′-CTCTGACAGAAGAAGCCAAGAAC-3′ 5′-GCTGTGTAGCATAGGGCAAG-3′
bFGF
ICAM-1
IL-8
NOS3
PDGFB
TGFβ
TSP-1
VCAM-1
VEGFA
5′-TGTGCTAACCGTTACCTGGCT-3′
5′-CTTCTCCTGCTCTGCAACCC-3′
5′-TGTGTGAAGGTGCAGTTTTG-3′
5′-GTGGCTGTCTGCATGGACCT-3′
5′-TTCCCGAGGAGCTTTATGAG-3′
5′-TCGCCAGAGTGGTTATCTTTT-3′
5′-CTTCAAGCCAGAGGCCTACG-3′
5´-CTGGAAATGCAACTCTCACC-3′
5´-ATCTTCAAGCCATCCTGTGTGC-3′
5′-CAGTGCCACATACCAACTG-3′
5′-GGGAGAGCACATTCACGGTC-3′
5′-ATTTCTGTGTTGGCGCAGT-3′
5′-CCACGATGGTGACTTTGGCT-3′
5′-GGGTCATGTTCAGGTCCAA-3′
5′-TAGTGAACCCGTTGATGTCC-3′
5′-CCGCCTCTGTCTTCTTCAGC-3′
5´-GTCTCCAATCTGAGCAGCAA-3′
5´-CAAGGCCCACAGGGATTTTC-3′
5′-CCATCCAATCGGTAGTAGCG-3′
18S rRNA 5′-GTAACCCGTTGAACCCCATT-3′
efficiency control. The mRNA fold change was calculated using the
method, with the values expressed as fold increases re-
lative to the untreated control (Livak and Schmittgen, 2001).
stage (annexin Vþ/PIꢀ) and late-stage (annexin Vþ/PIþ) apopto-
sis. No significant effect was observed in cell populations when
HUVECs were incubated with new TZDs or RSG for 24 h, sug-
ΔΔC(t)
2ꢀ
gesting 5
was the elected concentration for the following experiments.
Since PPAR is the main molecular target of TZDs, such as RSG
and pioglitazone, we examined whether the new TZDs might act
as agonists of PPAR using a reporter gene assay. As illustrated in
Fig. 2C, new TZDs induce PPAR activation. However, in contrast to
the full PPAR agonist RSG, only a weak activation of PPAR was
observed with new TZDs. Indeed, relative to full PPAR activation
μ
M as a nontoxic concentration (Fig. 2B). Hence, 5
μ
M
2.12. Data analysis
γ
All results are expressed as the mean7S.D. from triplicate
measurements performed in at least two independent experi-
ments. Statistical analysis was performed by one-way ANOVA
followed by Dunnett's test. Probability values were considered
significant at Po0.05.
γ
γ
γ
γ
γ
induced by RSG, the detected activation with new TZDs was ap-
proximately 20–25%, suggesting that these compounds might act
as partial agonists of PPAR .
γ
3. Results
3.1. New TZDs show low cytotoxicity on endothelial cells and are
weak PPARγ agonists
3.2. New TZDs reduce ROS production and increase intracellular le-
vels of nitric oxide in HUVEC
To determine the cytotoxicity of new TZDs, HUVEC were trea-
ted with vehicle (0.01% DMSO) or various concentrations (0.625–
To evaluate the effects of new TZDs on oxidative stress in
HUVEC, cells were incubated in normal and high-glucose medium,
co-treated with new TZDs or RSG for 24 h, and the intracellular
ROS production was examined using the probe CM-H2DCFDA. As
shown in Fig. 3A, a significant increase in CM-H2DCFDA-associated
fluorescence was observed in the cells incubated with 30 mM
glucose compared with 5.6 mM glucose medium. On the other
hand, the treatment with RSG, GQ-169, and LYSO-7 inhibited the
glucose-induced ROS production, whereas GQ-32 did not affect
intracellular ROS production in cells incubated with 30 mM glu-
cose. In addition, when HUVECs were incubated in normal glucose
condition (5.6 mM), neither RSG nor new TZDs affected ROS pro-
duction (Supplementary Data).
10 M) of new TZDs for 24 h, and cell viability was analyzed by
μ
MTT assay. As shown in Fig. 2A, similar to RSG, new TZDs show low
cytotoxicity on HUVEC. However, GQ-32 and GQ-169 induced a
significant decrease in cell viability at 10 M. In contrast, treat-
μ
ment with LYSO-7 did not alter the overall viability of the cells
compared with cells treated with vehicle.
Although a decrease of cell viability was also detected with GQ-
32 and GQ-169 at 5
lular surface were observed. Therefore, to further confirm 5
μ
M, no morphological alterations of the cel-
M as
μ
a nontoxic concentration, we evaluated the effects of new TZDs on
cell death. HUVECs were then incubated with new TZDs or RSG for
24 h, and cell death was determined with annexin V/PI staining by
flow cytometry evaluating four different populations: viable cells
(annexin Vꢀ/PIꢀ), nonviable cells (annexin Vꢀ/PIþ), and early-
The effects of new TZDs were also explored on production and
release of nitric oxide in HUVEC. As depicted in Fig. 3B, new TZDs
significantly increased intracellular levels of NO after 4 h
Fig. 2. New TZDs show low cytotoxicity in HUVECs and act as partial agonists of PPARγ. (A) The effects of new TZDs on cell viability were measured by MTT assay. HUVECs
were incubated with increasing concentrations of GQ-32, GQ-169, LYSO-7, or RSG for 24 h and then analyzed as indicated. Data are expressed as percentage of vehicle (0.01%
DMSO)-treated control culture conditions. N¼8 from three independent experiments. (B) Effects of new TZDs on endothelial cell death were determined with annexin/PI
staining by flow cytometry after incubation of HUVECs with new TZDs or RSG (at 5 μM) for 24 h. N¼6 from two independent experiments. (C) Cellular activation of PPARγ
was determined with a reporter gene assay. Human embryonic kidney 293 cells were transiently transfected with specific plasmids and treated with increasing con-
centrations of GQ-32, GQ-169, LYSO-7, or RSG for 16 h. Transcriptional responses were evaluated using a luciferase reporter gene assay kit. N¼6 from two independent
experiments. Data are expressed as mean7S.D. *Po0.05 vs. control group.

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M. Rudnicki et al. / European Journal of Pharmacology 782 (2016) 98–106
Fig. 3. New TZDs affect ROS and NO production in HUVECs. (A) Quantification of effects of new TZDs on ROS intracellular levels relative to control group. HUVECs were
incubated with RPMI 1640 medium (5.6 mM glucose or 30 mM glucose) plus a 5 μM concentration of new TZDs or RSG, and ROS intracellular levels were measured using the
probe CM-H2DCFDA. (B–C) New TZDs affect intracellular levels (B) and release (C) of NO. (B) HUVECs were incubated with new TZDs or RSG (at 5 μM) for 4 h, and intracellular
levels of NO were determined with NO-specific fluorescence probe DAF-FM. (C) Quantification of NO release relative to control group. NO release was measured with
chemiluminescence analysis of culture medium after treatment with new TZDs or RSG (at 5 μM) for 24 h. (D) HUVEC eNOS expression after treatment with new TZDs or RSG
for 4 h. The mRNA levels of eNOS were explored by real-time PCR. Data are expressed as mean7S.D. of three independent experiments performed at least in triplicate.
*Po0.05,**Po0.01,***Po0.001 vs. control group, #Po0.001 vs. control group incubated with 5.6 mM glucose medium.
incubation. Furthermore, NO release was augmented when HU-
VECs were treated with LYSO-7 or RSG for 24 h (Fig. 3C). On the
impact of new TZDs on migration in HUVECs using the in vitro
wound scratch assay. As demonstrated in Fig. 5A, GQ-32 and GQ-
169 significantly induced endothelial cell migration, whereas no
effect was observed with LYSO-7 treatment. It is noteworthy that,
other hand, treatments with 5 M GQ-32 and GQ-169 were unable
μ
to affect NO release (Fig. 3C). In addition, to determine whether
new TZDs regulate NO production through modulation of en-
dothelial nitric oxide synthase (eNOS) expression, HUVECs were
treated for 4 h, and mRNA levels of eNOS were determined with
real-time PCR. As can be observed in Fig. 3D, eNOS mRNA ex-
pression was not affected by new TZDs or RSG.
under our experimental conditions, 5 M RSG reduced endothelial
μ
cell migration by 16%, but this was not statistically significant
(Fig. 5B).
To further verify that new TZDs would also affect endothelial
capacity to form capillary-like structures, an in vitro tube forma-
tion assay was performed using Matrigel basement membrane
matrix, which mimics capillary formation in vivo. As illustrated in
Fig. 5C, RSG suppressed the formation of tube-like structures. In
contrast, LYSO-7 did not affect tube formation, whereas GQ-32 and
GQ-169 stimulated the formation of tube-like structures (Fig. 5C-
D).
3.3. New TZDs down-regulate TNF-
molecules in HUVECs
α-induced expression of adhesion
To evaluate the effects of new TZDs on endothelial cell activa-
tion, HUVECs were incubated with new TZDs for 4 h in the absence
or presence of 10 ng/ml TNF-
vels of proinflammatory adhesion molecules VCAM-1, ICAM-1, and
E-selectin were remarkably up-regulated in response to TNF- . As
expected, RSG significantly attenuated the TNF- -induced ex-
pression of VCAM-1, ICAM-1, and E-selectin. Similar to RSG,
treatment with 5 M GQ-32, GQ-169, and LYSO-7 suppressed the
induction of VCAM-1 and ICAM-1 stimulated by TNF- . In con-
trast, the TNF- -induced expression of E-selectin was unaffected
by the new TZDs (Fig. 4).
α
. As shown in Fig. 4, the mRNA le-
To understand the cellular and molecular mechanisms through
which new TZDs affected cell migration and tube formation of
endothelial cells, we evaluated the effects of new TZDs on gene
expression of angiogenic molecules. Consistent with increased cell
migration and formation of tube-like structures, GQ-32 and GQ-
169 induced vascular endothelial growth factor A (VEGFA) ex-
pression (Fig. 6A). The treatment with GQ-169 also up-regulated
the mRNA levels of basic fibroblast growth factor (bFGF, Fig. 6B)
and interleukin 8 (CXCL8, Fig. 6C) and did not affect transforming
α
α
μ
α
α
growth factor
duced expression of TGF
influence mRNA bFGF levels (Fig. 6B). RSG inhibited VEGFA mRNA
expression, and LYSO-7 down-regulated platelet-derived growth
factor (PDGF)-BB mRNA levels (Fig. 6E). Both LYSO-7 and RSG also
β
(TGF
β
(Fig. 6D)). expression. In turn, GQ-32 in-
(Fig. 6D) and CXCL8 (Fig. 6C) but did not
3.4. New TZDs affect endothelial cell migration and tube formation
in vitro
β
We next tested whether new TZDs would also affect biological
processes involved in angiogenesis. Because migration of en-
dothelial cells is crucial during angiogenesis, we explored the
decreased the expression of TGF
of thrombospondin-1, a potent inhibitor of angiogenesis, was not
β
mRNA (Fig. 6D). The expression

M. Rudnicki et al. / European Journal of Pharmacology 782 (2016) 98–106
103
vascular homeostasis and modulates the vascular tone, endothelial
cell growth, adhesion of platelet and leukocytes, platelet ag-
gregation (Lundberg et al., 2015) and angiogenesis (Ridnour et al.,
2005). It is noteworthy that reduced bioavailability of NO, the
main feature of endothelial dysfunction, is associated with the
pathogenesis of vascular complications and that strategies to in-
crease NO signaling have been extensively explored. Corroborating
results from previous studies (Calnek et al., 2003; Polikandriotis
et al., 2005), we show that RSG increases the production and re-
lease of NO without affecting eNOS expression. LYSO-7 demon-
strates a similar effect compared with RSG. However, GQ-32 and
GQ-169 display only a weak and transient effect on NO production
as both treatments failed to modify the release of NO from HUVEC
measured by chemiluminescence analysis of culture media.
Accumulating evidence indicates that increased ROS produc-
tion also may influence endothelial cell function and plays a cru-
cial role in the development of vascular pathologies (Schaffer et al.,
2012). In this respect, previous studies propose that antioxidant
agents contribute to the prevention of vascular complications. Of
note, the suppression of glucose-induced ROS production is de-
scribed as a beneficial effect of TZDs class of drugs (Fujisawa et al.,
2009). Therefore, we hypothesized that the new TZDs could affect
glucose-induced ROS production. Our results show that only GQ-
169 and LYSO-7 reduced glucose-induced ROS production whereas
GQ-32 failed to show an effect on ROS production in endothelial
cells under our experimental conditions.
TZD class of drugs also presents anti-inflammatory properties
not only in endothelial cells but also in different cell types and
animal models (Pasceri et al., 2000; Santin et al., 2013a; Wang
et al., 2002). The anti-inflammatory effects of TZDs have been
suggested to be mediated through suppression of adhesion mo-
lecules expression (Pasceri et al., 2000; Wang et al., 2002), which
regulate the recruitment and binding of circulating leukocytes to
the vessel wall and are essential in inflammatory responses (Golias
et al., 2007). As expected, in our study, RSG inhibited the expres-
sion of VCAM-1, ICAM-1, and E-selectin in response to TNF-
terestingly, although GQ-32, GQ-169, and LYSO-7 have poor ago-
nist activities on PPAR compared to RSG, they show equivalent
inhibition of TNF- -induced expression of VCAM-1 and ICAM-1,
suggesting that the inhibitory effects on endothelial cell activation
of these new TZDs may be PPAR independent. In fact, previous
studies employing PPAR null cells, demonstrate that PPAR is not
α
. In-
γ
α
γ
γ
γ
essential for the anti-inflammatory effects of TZDs (Chawla et al.,
2001; Feinstein et al., 2005). Our results are also in agreement
with recent data describing the anti-inflammatory effects of LYSO-
7. LYSO-7 has been also shown as a cyclooxygenase inhibitor, and
its anti-inflammatory effects have been already established in
neutrophils (Santin et al., 2013b) and macrophages, as well as in a
mouse model of atherosclerosis (Cesar et al., 2015).
Fig. 4. Effects of new TZDs on mRNA levels of adhesion molecules. HUVECs were
incubated with new TZDs or RSG at 5 μM and cotreated with TNF-α (10 ng/ml) for
4 h. The mRNA levels of VCAM-1 (A), ICAM-1 (B), and E-selectin (C) were explored
by real-time PCR. Data are expressed as mean7S.D. of three independent experi-
ments performed in triplicate. #Po0.001 vs. control group without TNF-α,*Po0.05,
**Po0.01,***Po0.001 vs. control group with TNF-α.
Previous studies exploring the effects of TZDs on angiogenesis
have shown controversial results. Evidence from in vitro and
changed either by RSG or by new TZDs (Fig. 6F).
in vivo models indicate that, through PPAR activation, TZDs might
γ
demonstrate antiangiogenic effects (Aljada et al., 2008; Panigrahy
et al., 2002; Xin et al., 1999), but also promote angiogenesis, which
was associated with eNOS activation and resulted in enhanced
neovascularization (Biscetti et al., 2008; Chu et al., 2006; Gealek-
man et al., 2008; Nagahama et al., 2012). Discrepancies between
these results probably rely on study design, PPAR affinity/activa-
4. Discussion
In this study, we report that three new TZDs, namely GQ-32,
GQ-169, and LYSO-7 affect endothelial cell functions and activa-
tion. We show that the new TZDs act as partial agonists of PPAR ,
γ
inhibit ROS production and adhesion molecules expression, and
influence NO production. We further reveal that GQ-32 and GQ-
169 also stimulate endothelial cell migration and tube formation in
vitro. Interestingly, compared with RSG, LYSO-7 demonstrates
equivalent properties on HUVEC, whereas the evidence presented
here supports that GQ-32 and GQ-169 possess similar anti-in-
flammatory properties, but distinct effects on angiogenesis.
Endothelium-derived NO is a signaling molecule relevant to
tion and PPAR -independent actions of TZDs. Under our experi-
γ
mental conditions, the new TZDs, GQ-32 and GQ-169, stimulate
two hallmarks of angiogenesis, endothelial cell migration and their
differentiation into tube-like structures. By contrast, in line with
previous studies, we also showed that RSG inhibited the en-
dothelial tube formation under our experimental conditions,
strengthening the antiangiogenic action associated with this TZD.
A
major limitation of our study is that the underlying

104
M. Rudnicki et al. / European Journal of Pharmacology 782 (2016) 98–106
Fig. 5. Effects of new TZDs on angiogenesis. (A) A cell-free area was introduced with a pipette tip in HUVEC confluent monolayers, and cells were incubated with RPMI
1640, 1% FBS containing 5 μM GQ-32, GQ-169, LYSO-7, or RSG for 24 h. Representative pictures show cell migration after 24 h of treatment. Dashed lines indicate the initial
scratch margins. (B) Quantification of HUVEC migration relative to control group. Data are expressed as mean7S.D. *Po0.05 vs. control group. (C) Tube formation of HUVECs
using Matrigel basement membrane matrix. Representative photographs after 6-h treatment with new TZDs and RSG. (D) Graphical representation of tube formation. Total
tube length as calculated by the ImageJ angiogenesis analyzer. Data are expressed as mean7S.D. *Po0.05 vs. control group.
mechanisms responsible for the proangiogenic effects of GQ-32
and GQ-169 were not completely elucidated. However, since an-
giogenesis is regulated by the balance of proangiogenic and anti-
angiogenic molecules (Carmeliet and Jain, 2011), we speculate that
the enhanced expression of proangiogenic molecules might con-
tribute to the effects of new TZDs on angiogenesis. Our results
reveal that GQ-32 and GQ-169 increased the mRNA levels of
VEGFA in HUVEC, whereas an opposite effect was observed with

M. Rudnicki et al. / European Journal of Pharmacology 782 (2016) 98–106
105
Fig. 6. Effects of new TZDs on mRNA levels of angiogenic molecules. HUVECs were incubated with new TZDs or RSG at 5 μM for 4 h, and the mRNA levels of VEGFA (A),
bFGF (B), CXCL8 (C), TGFβ (D), PDGF-BB (E), and thrombospondin-1 (TSP-1, F) were explored by real-time PCR. Data are expressed as mean7S.D. of three independent
experiments performed in triplicate. *Po0.05,**Po0.01,***Po0.001 vs. control group.
RSG. VEGFA is the major modulator of angiogenesis and is involved
in the regulation of endothelial cell proliferation, survival, and
migration (Koch and Claesson-Welsh, 2012). CXCL8, bFGF, PDGF-
32, GQ-169, and LYSO-7 act as partial agonists of PPAR and show
γ
beneficial pleiotropic effects, affecting endothelial cell functions
and activation. In addition, the up-regulation of proangiogenic
molecules as well as increased cell migration and tube formation
observed with GQ-32 and GQ-169 suggest a therapeutic potential
in the treatment of clinical conditions associated to impaired an-
giogenesis, such as ischemic conditions and impaired wound
healing.
BB, and TGF
β
are also described as relevant molecular players
involved in angiogenesis (Carmeliet and Jain, 2011). The evidence
demonstrates that bFGF and CXCL8 modulate endothelial cell mi-
gration and capillary tube formation (Li et al., 2005) and PDGF-BB
and TGF contribute to the vessel maturation (Carmeliet and Jain,
β
2011; Pardali et al., 2010). In our study, although both GQ-32 and
GQ-169 enhanced CXCL8 expression, only the treatment with GQ-
169 up-regulated the mRNA levels of bFGF. GQ-32 also increased
Conflict of Interest
the expression of TGF , whereas decreased mRNA levels were
β
observed with RSG and LYSO-7. Thus, GQ-32 and GQ-169 induce
angiogenesis and increase the gene expression of proangiogenic
molecules, thereby revealing that these new TZDs might have
different biological actions and activities when compared to full
None declared.
Acknowledgments
PPAR
γ
agonists.
Taken together, these findings suggest that the new TZDs GQ-
This study was supported by São Paulo Research Foundation

106
M. Rudnicki et al. / European Journal of Pharmacology 782 (2016) 98–106
(FAPESP Grant 2012/51316-5 to D.S.P.A.) and National Council for
Scientific and Technological Development [National Institute of
Science and Technology for Pharmaceutical Innovation ((INCT_if/
CNPq) Grant 573663/2008-4 to I.R.P.] and CNPq/MICCIN Grant
BFU2011-2476 to D.S.P.A. and L.B.G.). R.F. was supported by FAPESP
Fellowship 2011/24020-5, and M.R. was supported by fellowships
from the FAPESP (2009/53072-3) and the CNPq (151568/2013-8).
Endocrinol. Metab. 295, E1056–E1064.
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