Hindawi
PPAR Research
Volume 2019, Article ID 2715176, 9 pages
https://doi.org/10.1155/2019/2715176

Research Article
Peroxisome Proliferator-Activated Receptor-𝛾
Antagonizes LOX-1-Mediated Endothelial Injury by
Transcriptional Activation of miR-590-5p
Lei Xu,1 Gang Zhao,1 Hong Zhu,1,2 Shijun Wang ,1 Aijun Sun,1
Yunzeng Zou,1 and Junbo Ge 1
1

Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China
Laboratory of Oral Microbiology, Shanghai Research Institute of Stomatology, Shanghai Key Laboratory of Stomatology, Ninth People's
Hospital, School of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China

2

Correspondence should be addressed to Shijun Wang; wang.shijun@zs-hospital.sh.cn and Junbo Ge; jbge@zs-hospital.sh.cn
Received 6 March 2019; Revised 7 May 2019; Accepted 27 May 2019; Published 1 July 2019
Academic Editor: Nanping Wang
Copyright © 2019 Lei Xu et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is one of the major receptors expressed on the endothelium ofarterial wall with a key role in endothelial dysfunction and the development of atherosclerosis. Recent evidence suggested that
LOX-1 is upregulated under the condition of insulin resistance and could be suppressed by the antidiabetic drugs. We previously
also confirmed that Thiazolidinedione (TZD) has the inhibitory effect on LOX-1 in ox-LDL-induced endothelial cells. However,the underlying mechanism is unclear. Here we showed that Rosiglitazone treatment significantly attenuated the expressions of
LOX-1, ICAM-1, VCAM-1, p47phox , and the atherosclerotic lesions in ApoE−/− mice with high-fat diet. In vitro, we revealed that
Rosiglitazone inhibited LOX-1 by regulating miR-590-5p. Ox-LDL-mediated ICAM-1, VCAM-1, and p47phox were significantly
reduced by Rosiglitazone, but all reversed after pretreating the cells with antagomiR-590-5p. Induction with Rosiglitazone activated
PPAR-𝛾 and promoted its nuclear translocation in cultured human umbilical vein endothelial cells (HUVECs). The nuclear PPAR𝛾 upregulated the miR-590-5p level through binding to its transcriptional promoter region. Retaining PPAR-𝛾 in cytoplasm bytransfecting with PPAR-𝛾RNLS plasmid in HUVECs failed to activate miR-590-5p. Mutation of the promoter region of PPAR-𝛾
also reduced the miR-590-5p promoter luciferase activity. Collectively, these data indicated that PPAR-𝛾 may have the therapeuticpotential in atherosclerosis via the transcriptional regulation of miR-590-5p in endothelial cells.

1. Introduction
Endothelial oxidative injury is considered as the leading
cause of coronary atherosclerosis, and oxidized low-density
lipoprotein cholesterol (ox-LDL) is critically involved inendothelial oxidative injury and dysfunction [1]. Lectinlike oxidized low-density lipoprotein receptor-1 (LOX-1) is
the major scavenger receptor for Ox-LDL, which promotes
the uptake of ox-LDL by endothelial cells (ECs) in thearterial wall, and accelerates the process of inflammation andoxidative stress [1, 2]. The basal line of LOX-1 in ECs is very
low, but it can be rapidly induced by prooxidative stress, suchas ox-LDL, angiotensin II (AngII), advanced glycation end
productions (AGEs), and proinflammatory cytokines [2–4].

The activation of LOX-1 promotes EC uptake of large amountsof ox-LDL, leads to apoptosis of vascular endothelial andsmooth muscle cells, increases the production of matrix metalloproteinases and intercellular adhesion molecules, which
promotes migration and infiltration of inflammatory cells,
and accelerates the atherosclerotic progression and plaquevulnerability [2, 5].
Peroxisome proliferator activated receptors (PPARs)
belong to the superfamily of ligand-activated transcription
factors, including PPAR-𝛼, PPAR-𝛽/𝛿, and PPAR-𝛾 threesubtypes [6, 7]. Different from the others, PPAR-𝛾 is mainly
expressed in adipocytes and vascular endothelial cells [8].
The wide spectrum effects of PPAR-𝛾 activation may bebeneficial for lipid metabolism, promoting free fatty acid2𝛽-oxidation, reducing the accumulation of plasma triglycerides, and preventing the development of atherosclerosis[7, 9]. Abnormal fat distribution and partial lipodystrophy
are observed in mutant PPAR-𝛾 gene patients [10]. In ourprevious work, we showed that ox-LDL-mediated endothelial
LOX-1 upregulation was suppressed by Ciglitazone, a special
PPAR-𝛾 agonist. However, the underlying mechanism isunclear.
Previous studies indicated that ox-LDL-induced LOX1 activation could be inhibited by miR-590-5p [11, 12], andmiR-590-5p agomir effectively prevented the development of
atherosclerosis in ApoE−/− mice through attenuating lipid
accumulation and proinflammatory cytokine secretion [13,14]. Induction with miR-590-5p effectively decreased cellular reactive oxygen species (ROS), blocked the p38MAPK
signaling pathway, and inhibited the nuclear translocationof Nf-𝜅B by targeting LOX-1 [9, 12]. Emerging lines ofevidence suggested that PPAR-𝛾, as an important nuclear
transcriptional factor, promoted the transcription of multiple
microRNAs including miR-125a, miR-424, and miR-503,
which were critically involved in regulating endothelialdependent inflammation and angiogenesis [15, 16]. Therefore,
in this study we hypothesized that PPAR-𝛾 activation might
inhibit ox-LDL-induced LOX-1 by targeting miR-590-5p,
which improved and even reversed the pathological courseof atherosclerosis.

PPAR Research
2.3. In Vitro HUVECs Culture and Cell Immunostaining.
Primary HUVECs were purchased from AllCells (Shanghai,
China) and cultured in low-glucose DMEM with 10% FBS(Gibco). After being in vitro cultured and passaged for 8∼10
times, the cells were used for experiments. After treating thecells with or without GW9662 (5 𝜇mol/L) for 1 hour followedby the induction with Rosiglitazone (50 𝜇mol/L) for 24 hours,
HUVECs were fixed by 4% paraformaldehyde, permeabilized
in 0.1% Triton X-100, and blocked with 3% BSA for 30 min,and then they were stained by anti-PPAR-𝛾 (1:200, ab45036,
Abcam) for 1 hour at 4∘ C overnight. After washing for 3 times,
the cells were stained by Alexa Fluor 488-conjugated goatanti-rabbit IgG (1:1000).
2.4. Reconstruction and Transfection of the Plasmids. The
mutant PPAR-𝛾 without the NLS peptide (PPAR-𝛾RNLS )was performed by plasmid reconstruction. The NLS peptide
with a core residue “LSVMDDHSH” located at the junction
between DNA binding domain (DBD) and hinge domain of
PPAR-𝛾 gene was conserved in different species [19, 20]. Thisregion was deleted in the wild-type (WT) PPAR-𝛾 plasmid(Obio Tech, Shanghai) using restriction endonuclease and
PCR fragment amplification method. The linear plasmid
was recombined with T4 DNA ligase. Both WT PPAR𝛾 and PPAR-𝛾RNLS were transfected into HUVECs using
Lipofectamine LTX with Plus reagent (Life Technologies)
according to the manufacturer’s instructions.

2. Methods
2.1. Animal Model and Sample Collection. Eight-week oldmale C57BL/6 ApoE gene knockout (ApoE−/− ) mice werepurchased from Beijing Vital River Laboratory Animal
Technology Co., Ltd. All the mice were housed in SPF
conditions and randomly divided into three groups, thecontrol group (n=10), the group of ApoE−/− mice (n=10)
which only received high-fat diet, and another group (n=10)
which received high-fat diet with Rosiglitazone (2mg/kg/day,
BRL49653, MCE) by oral gavage daily, respectively, for 10
weeks as previously described [17]. Body weights of all themice were monitored and recorded for every week. After
10 weeks, all the mice were sacrificed by inhaled anesthesia
with 3% isoflurane. The blood sample of each mouse collectedby extracting the eyeball blood and the plasma levels of TC
and HDL-C were determined by enzymatic method using
commercial kits as previously described [18]. The aortas andrelated tissues were isolated from mice and were quickly fixed
for immunohistochemistry slice or frozen in liquid nitrogenfor protein analysis.
2.2. Quantiﬁcation of Atherosclerotic Lesion Areas. After themice were sacrificed, the chest was opened and the proximal
aorta attached to the heart was isolated and fixed in 4%
paraformaldehyde for 24 h. Cross-section (∼7 𝜇m) of theaorta root was collected and stained with oil-red O andbrilliant green. The volume of atherosclerotic lesions wascalculated from five different sections. Quantitative analysisof stained lipid area was performed by a blinded observerusing Leica QWin V3 software.

2.5. Western Blot Analysis. HUVECs grown on six-well plate
were lysed in RIPA buffer containing the protease inhibitor,
1mM PMSF (ST506, Beyotime Biotech.) on ice for 20 min.
Total proteins were extracted from the lysates by centrifugation (11000 rpm, 4∘ C, 10 min). Then the proteins (20 𝜇g)
were loaded onto SDS-PAGE gels (12%) and separated byelectrophoresis, followed by transfer onto the PVDF membrane (Bio-Rad). The immunoblots on the membrane were
blocked with 5% BSA and then incubated with the primary
antibodies (anti-LOX-1, rabbit polyclonal antibody at 1:1000
dilution, ab203246, Abcam; anti-ICAM-1, mouse monoclonal
antibody at 1:1000 dilution, ab20, Abcam; anti-VCAM-1,
rabbit monoclonal antibody at 1:1000 dilution, ab 174279,
Abcam; and anti-p47phox , rabbit polyclonal antibody at 1:1000
dilution, #4312, Cell Signaling Technology). After washingwith 1×PBST for 3 times, the blots were incubated again
with the horseradish peroxidase (HRP) conjugated secondary
antibody IgG (1:5000, Kangchen Biotechnology, Shanghai).
GAPDH was used as the internal control. The immunoblot
complexes were detected by high resolution chemiluminescence Western blot detection reagents (Thermo scientific,
34080) and the images were captured by ChemiDocTM
Touch Imaging System (BIO-RAD, CA, USA).
2.6. Chromatin Immunoprecipitations (ChIPs). Aftertransfecting the WT-PPAR-𝛾 or PPAR-𝛾RNLS plasmid into
HUVECs, the cells were stimulated by Rosiglitazone(50 𝜇mol/L) for 24 h. Cells were then fixed with 1%
formaldehyde for 10 min and stopped by adding glycine
to a final concentration of 200 mmol/L. After washing

PPAR Researchwith ice-cold 1×PBS for 3 times, cells were homogenizedon ice by a sonicator within cell lysis buffer containing
0.2% NP-40 and protease inhibitors. Then, chromatin
DNA were extracted and sheared into ∼500 bp fragments.
After centrifugation for 5 min, the supernatant was diluted
with 10-fold dilution buffer and reserved as negative
control. The remaining lysates were precleared with
Dynabeads Protein A (50 𝜇L) and agitated at 4∘ C for 30
min. The anti-PPAR-𝛾 antibody (1:200, ab45036, Abcam)
or a control IgG (1:200) was added into the precleaned
supernatant, and then the supernatant was incubated at
4∘ C overnight with constant rotation. ChIP beads were
washed six times at room temperature and incubatedwith proteinase K for 30 min and reverse cross-linked
by heating at 65∘ C for 4 hours. The immunoprecipitated
DNA was purified by phenol/chloroform extraction andrecovered for PCR reaction, and the temperature cycling
was denaturation at 95∘ C for 30s, annealing at 56∘ C for 30s,
and extension at 72∘ C for 30s, and it was repeated for 34
cycles. PCR primers were as follows: the upstream primer:
5󸀠 -CCTCTCCTTCCCCTTCTCCT-3󸀠 ; the downstream
primer: 5󸀠 -TTAAAGGCTGAACACGGTGG-3󸀠 .
2.7. Luciferase Report Assay. Promoter luciferase reporter
assay was produced by a pMIR-REPORT system as
described previously [21]. The miR-590-5p promoter regions
containing the PPAR-𝛾 core binding motif “TAGGTCA” or
the mutated type (mut) motif “TAAATAA” were, respectively,
amplified and cloned into the pMIR-REPORT Luciferase
plasmid, which located at the upstream of the luciferase
reporter gene and was driven by the CMV enhancer. Each
type of reporter plasmids and pCMV-Renilla control were,respectively, transfected into HEK-293T cells 48 h before
stimulating with Rosiglitazone. Cell lysates were collected
and the luciferase activities were assayed by Dual-Luciferase
Reporter Assay System (Promega, Madison, WI) according to
the manufacturer’s instructions.
2.8. Statistical Analysis. All data are presented as mean ±SEM. Analyses were performed with statistical software SPSS
16.0. The differences among groups were analyzed by OneWay ANOVA of variance followed by LSD analysis. Values
were considered statistically significant when P < 0.05.

3. Results
3.1. Rosiglitazone Suppressed Endothelial LOX-1 in Atherosclerotic ApoE−/− Mice. To investigate the role of PPAR-𝛾 activation in regulating endothelial function in an atherosclerosis
model, the ApoE−/− mice were subjected to high-fat diet for
10 weeks with or without Rosiglitazone. The results showedthat Rosiglitazone treatment reduced the body weight gain of
ApoE−/− mice by 15.2% (Figure 1(a)). Compared with highfat-induced ApoE−/− mice, plasma levels of TC (721.8±130.4
vs. 2254.3±276.0 mg/dL, P <0.05) and HDL-C (101.3±11.5 vs.
246.5±26.1mg/dL, P <0.05) were both significantly reducedin Rosiglitazone-pretreated mice (Figures 1(c) and 1(d)).
Consistent with the abnormal elevation of plasma TC and

3
HDL-C, severe atherosclerotic plaques were observed in
the aortic root of coronary artery of ApoE−/− mice, but
the atherosclerotic lesions were not obviously augmented in
Rosiglitazone pretreated ApoE−/− mice (Figure 1(b)). Of note,the increased protein level of LOX-1 in the coronary endothelium of ApoE−/− mice after high-fat diet was significantly
decreased by Rosiglitazone. Accompanied by the upregulation of LOX-1, high-fat diet also increased the expressions of ICAM-1, VCAM-1, and p47phox in ApoE−/− mice,
which were all reversed by Rosiglitazone treatment (Figures
1(e)–1(h)), suggesting that PPAR-𝛾 activation might effectively suppress LOX-1 expression and prevent endothelial
inflammation and oxidative injury during the development of
atherosclerosis.
3.2. miR-590-5p Was Downregulated in ox-LDL-Induced
HUVECs. Previous studies indicated that LOX-1 could be
negatively regulated by miR-590-5p; therefore we determinedthe expression of miR-590-5p in ox-LDL-induced humanumbilical vein endothelial cells (HUVECs). As shown in
Figure 2, the baseline level of miR-590-5p was high in in vitro
cultured HUVECs, but it gradually decreased in HUVECs
after stimulating by dose-dependent ox-LDL (Figure 2(a)).
To confirm the role of miR-590-5p in regulating LOX-1,
we transfected the miR-590-5p expression plasmid (pMIRCMV-miR590, Vigene Bioscience) into HUVECs and thenexamined the cellular LOX-1 after stimulating the cells with
ox-LDL (20 𝜇g/ml) for 24 h. Compared with the effectof control plasmid, miR-590-5p overexpression prevented
the upregulation of LOX-1 in ox-LDL-induced HUVECs(Figures 2(b) and 2(c)). In addition, ICAM-1, VCAM-1, andp47phox were all suppressed in ox-LDL-induced HUVECs
transfecting by miR-590-5p expression plasmid (Figures 2(b),2(d), 2(e), and 2(f)). Importantly, the level of miR-590-5p
in HUVECs repressed by ox-LDL was rebounded after pretreating the cells with Rosiglitazone (Figure 2(g)). To determine whether PPAR-𝛾 activation inhibited LOX-1 throughregulating miR-590-5p, we suppressed the expression of miR590-5p in HUVECs by introducing antagomiR-590-5p. Compared with the effect of antagomiR-NC, antagomiR-590-5p
pretreatment maintained ox-LDL-induced LOX-1 expressionin Rosiglitazone-pretreated HUVECs (Figures 2(h) and 2(i)),
and Rosiglitazone also failed to suppress ox-LDL-induced
ICAM-1, VCAM-1, and p47phox in HUVECs after blocking
miR-590-5p (Figures 2(h), 2(j), 2(k), and 2(l)), indicating thatmiR-590-5p is required for Rosiglitazone-mediated suppression of LOX-1 and LOX-1 mediated endothelial injury.
3.3. Rosiglitazone Upregulated miR-590-5p in HUVECs by
Activating PPAR-𝛾 and Promoting Its Nuclear Translocation. Next, we investigated whether PPAR-𝛾 activation isessential to Rosiglitazone-mediated upregulation of miR590-5p in endothelial cells. In vitro cultured HUVECs werepretreated with or without PPAR-𝛾 antagonist GW9662 (5𝜇mol/L) for 1 h and followed by Rosiglitazone treatment for
24 h, then the expression of miR-590-5p was detected incells. The results showed that miR-590-5p level significantly
increased in HUVECs with Rosiglitazone, but it did not

4

PPAR Research
35∗

Body weight (g)

30
25
20
15
10
5
0
ApoeE-/-(HF)

Apoe％-/- (HF)

ApoeE-/-(HF) + Rog

Apoe％-/- (HF) + Rog

(a)

(b)

LOX-1

58 kDaICAM-1

81 kDa

VCAM-1

47 kDap47ＪＢＩＲ

37 kDa

GAPDH

300

2000
∗

1000

250
200
150

∗

Co
nt
ro
l
H
ig
h
H
f
ig
at
h
di
fa
et
td
iet
+
Ro
g

plasma HDL-C (mg/dL)

3000

100
50

0

0
ApoeE-/- (HF)

ApoeE-/- (HF)

ApoeE-/- (HF) + Rog(c)

ApoeE-/- (HF) + Rog

(d)

(e)
0.4
Fold change p47/GAPDH

0.3
∗

0.2

0.1

(g)

Ro

g

iet

iet

+

fa
td
h

fa
td

h

h

H
ig

H
ig

H
ig

(f)

H
ig

iet

+

fa
td

Ro

g

iet

0

fa
td

fa
td

0

h

H
ig

h

iet

+

fa
td

Ro

iet

g

0

0.1

h

0.2

∗

0.2

H
ig

0.4

0.3

Co
nt
ro
l

∗

Fold change VCAM-1/GAPDH

0.6

Co
nt
ro
l

Fold change ICAM-1/GAPDH

0.8

Co
nt
ro
l

plasma TC (mg/dL)

31 kDa(h)Figure 1: Preferential induction of Rosiglitazone prevented atherosclerosis in high-fat diet-induced ApoE-/- mice. Male ApoE−/− mice were
subjected to high-fat diet with or without Rosiglitazone (2mg/kg/day) for continuous 10 weeks. (a) The body weight of mice in each group(n=10) was recorded at the end of 10 weeks. (b) Representative images of the cross-sectional aortic sinus stained with Oil Red O (scale bar,
100 𝜇m). (c) The plasma levels of total cholesterol (TC) and (d) high density lipoprotein cholesterol (HDL-C) were determined and comparedin both groups (∗ P<0.05). (e) Representative images of protein blots of LOX-1, ICAM-1, VCAM-1, and p47phox in aortic endothelium of the
ApoE−/− mice by Western blot. (f–h) Quantitative analysis of the protein levels of LOX-1, ICAM-1, VCAM-1, and p47phox (n=4, ∗ P<0.05 vs.
High fat diet group).

increase after being pretreated with GW9662 (Figure 3(a)).
In addition, we found that both cytoplasm and nuclear
PPAR-𝛾 were markedly enhanced in HUVECs after stimulating with Rosiglitazone, but the nuclear translocation of
PPAR-𝛾 attenuated in GW9662 pretreated HUVECs (Figures
3(b) and 3(c)). The effect of GW9662 on blocking PPAR𝛾 nuclear translocation was also confirmed by immunostaining (Figure 3(d)). To further confirm the function of
PPAR-𝛾 nuclear translocation in regulating miR-590-5p, wetransfected the WT or the mutant type of PPAR-𝛾 plasmid (PPAR-𝛾RNLS ), which lacked the nuclear localization
signal (NLS), respectively, into HUVECs (Figure 3(e)). In
HUVECs transfected by WT-PPAR-𝛾, miR-590-5p expression increased by 2-fold (n=3, P < 0.01), compared with thattransfected by PPAR-𝛾RNLS after stimulating with Rosiglitazone (Figure 3(e)), suggesting that the nuclear localizationof PPAR-𝛾 is critically involved in Rosiglitazone-mediatedactivation of miR-590-5p.

PPAR Research

51
31 kDaLOX-1

58 kDaICAM-1

81 kDa

VCAM-1

47 kDap47ＪＢＩＲ

37 kDa

GAPDH

0.6

0.4

0.2

l
ro
nt
Co

50

l
tro
on

L
LD

c
V-

-L
ox

+
DL
-L
ox

ox

CM
+p
DL

Fold change p47/GAPDH

0.2

(d)

(e)

90
m
iR
-5

on
tro
l

ox
-L
D
L

Co
nt
ro
l

m
VM
pC

LD
L+
p

+
DL
-L
ox

#
0.4

ox
-

M

pC

L+
LD

ox

∗

0

0

90

-5
iR

ox
-L
D
L+

l
ro
nt
co
V-

L
LD

ox

0.2

Co
nt
ro
l

l
ro
nt
Co

DL
-L
ox

m
VCM
+p

0.6

Vm
iR
-5
90

0

L
LD

CM

0.3

#

ro
l

#

0.8
∗

0.4

LD
L

0.6

90

-5
iR

c
V-

CM
+p

(c)

pC
M
Vco
nt

∗

l
tro
on

L
LD

ox

ox

0.6

ox
-

0.9

l
ro
nt
Co

(b)
Fold change VCAM-1/GAPDHFold change ICAM-1/GAPDH

(a)

0

90
-5
iR

m
VM
pC

Vc

20

CM

10

0.3

ox
-L
D
L+
pC
M
V-

5

#
0.6

D
L+
p

0

0.9

ox
-L

ox-LDL 0(g/ml)∗1.2
Fold change LOX-1/GAPDH

Relative miR-590-5p level

0.8

(f)

0.8

31 kDaLOX-1

58 kDaICAM-1

81 kDa

VCAM-1

47 kDap47ＪＢＩＲ

37 kDa

GAPDH

#
0.6
0.4

∗

Fold change LOX-1/GAPDH

Relative miR-590-5p level

1

0.2
0
Control

ox-LDL

1.2
0.9

$

0.6
#
0.3
0

ox-LDL
Rosiglitazone

+

+

+

+

-

+

+

+

ox-LDL++++

AntagomiR-590

-

-

+
-

-

Rosiglitazone

-

+

+

+

+

AntagomiR-590

-

-

+

-

AntagomiR-NC

-

-

-

+

ox-LDL+Rog

AntagomiR-NC

(g)

(i)

(h)

1.2

0.9
$
0.6
#

0.3
0

1

1.2

0.8

Fold change p47/GAPDHFold change VCAM-1/GAPDHFold change ICAM-1/GAPDH

1.5

$

0.6
0.4

#

0.2

$

1
0.8
0.6

#

0.4
0.2

0

0

+

ox-LDL++++

ox-LDL

+

+

+

+

+

Rosiglitazone

-

+

+

+

Rosiglitazone

-

+

+

+

+

-

AntagomiR-590

-

-

+

-

AntagomiR-590

-

-

+

-

-

+

AntagomiR-NC

-

-

-

+

AntagomiR-NC

-

-

-

+

ox-LDL+++

Rosiglitazone

-

+

+

AntagomiR-590--

AntagomiR-NC

-

-

(j)

(k)

(l)

Figure 2: miR-590-5p is required for the suppression of LOX-1 and LOX-1-dependent inﬂammatory and oxidative injury in HUVECs inducedby ox-LDL. (a) The expressions of miR-590-5p were determined in HUVECs stimulated by ox-LDL at a range of doses (0∼50 𝜇g/mL). (b–f)
The pMIR-CMV-miR590 and control plasmid were, respectively, transfected into HUVECs 48 h before the cells were stimulated by 20 𝜇g/mL
ox-LDL; the protein levels of LOX-1, ICAM-1, VCAM-1, and p47phox were detected and quantified by Western blot analysis. (g) The expressionof miR-590-5p in HUVECs stimulated by ox-LDL with or without Rosiglitazone (50 𝜇mol/mL). (h–l) The effect of antagomiR-590-5p and thenegative control (antagomiR-NC) on Rosiglitazone-induced protein levels of LOX-1, ICAM-1, VCAM-1, and p47phox in HUVECs stimulatedby ox-LDL (n=4, ∗ P<0.05; vs Control group; n=4, # P<0.05 vs. ox-LDL-induced group; $ P<0.05 vs. ox-LDL together with Rog treatedgroup).

3.4. Nuclear PPAR-𝛾 Activates the Transcription of miR-5905p through Binding to Its Promoter. The involvement of
PPAR-𝛾 in regulating miR-590-5p expression prompted us
to think whether miR-590-5p is one of the transcriptionaltargets of PPAR-𝛾. Therefore, we screened the location of
miR-590-5p in miRBase database (http:mirbase.org/). In the
promoter region (-2000bp∼-100bp) of miR-590-5p, we found

one predictable sequence-specific binding site for PPAR-𝛾(TAGGTCA) locating at -410∼-390 bp of the upstream of
miR-590-5p by JASPAR computational program database(Figure 4(a)). To test this hypothesis, we performed chromatin immunoprecipitation (ChIP)-PCR assay to examine protein-DNA interactions at the miRNA promoters in
HUVECs transfer with WT-PPAR or PPAR-𝛾RNLS plasmids.

PPAR Research

1.5∗

cytoplasma

1.2

nucleus

58 kDaPPARγ

58 kDaPPARγ

37 kDa

GAPDH

17 kDaHistone H3

GW9662

62
96

g

W
G

W

Ro

Co
nt
ro
l

96

g
G

g+

Rog

Ro

Control

g+

0

Ro

0.3

Ro

0.6

62

0.9

Co
nt
ro
l

Relative miR-590-5p level

6

(b)

(c)

(a)

Control

Rog

Rog+
GW9662

PPAR-γDAPI

Merged
(d)

Mouse
Rat
Human
MonkeyHinge

Relative miR-590-5p level

DBD
3
2.5
2
1.5
1
0.5
0

∗
∗

ol
ntr
Co

Ro

#

g
Ro

g

-γ
AR
P
+P
Ro

g

γ
RPA
P
+

LS
RN

(e)

Figure 3: miR-590-5p transcription is regulated by nuclear translocated PPAR-𝛾. Preferential induction the HUVECs with or without GW9662(5 𝜇mol/L) for 1 h followed by the induction with Rosiglitazone (50 𝜇mol/L) for 24 h and then determined (a) the expression of miR-590-5p,(b) the cytoplasm PPAR-𝛾 protein level, and (c) the nucleus PPAR-𝛾 protein level and (d) immunofluorescent staining of HUVECs using antiPPAR-𝛾 antibody (green) and dapi staining (purple DNA dye). (e) The expressions of miR-590-5p were determined in HUVECs induced by
Rosiglitazone and Rosiglitazone-induced HUVECs transfected with PPAR-𝛾 or PPAR-𝛾RNLS plasmid lacking the nuclear localization signal(NLS) (n=3, ∗ P<0.05; vs. Control group; # P<0.05 vs. PPAR-𝛾 with Rog treated group).

PPAR Research7

miR-590 promoter
PPAR-γ187bp

SV40 polyA(6438)
SV40 Promoter(6139)(6046)

NotI (407)(1)

WT : 5’-NNNTAGGTCANNN-3’
mut : 5’-NNNTAAATAANNN-3’
Insersion site

Puromycin(5447)(5188)
SV40 pA(5153)
Sspl(4961)(4943)

Luciferase

pMIR-REPORTTM

Luciferase

EcoRI (1609)

PP
AR

，３

(b)

Relative Luciferase Activity

(a)

PP
AR
- R
．

Ig
G

N
C

-390

In
pu
t

-410

1.6

∗

1.4
1.2
1
0.8
0.6
0.4
0.2
0

6470 bp

WT-PPAR promoter

Ampicillin

mut-PPAR promoter

BamHI (2205)(4083)(4024)
CoIE1 Origin

CMV Promoter(3138)

EcoRI (2808)(c)

PBS control
Rosiglitazone(d)Figure 4: Nuclear PPAR-𝛾 activates the transcription of miR-590-5p through binding to its promoter. (a) A schematic diagram depicting
the binding sites for PPAR-𝛾 in the promoter of miR-590-5p. (b) ChIP assays were performed with PPAR-𝛾 antibody and the miR-5905p promoter region with the PPAR-𝛾 binding motif was amplified by PCR. (c) A schematic representation of the insertion of WT-PPAR-𝛾 or
mut-PPAR-𝛾 DNA binding motif, locating at miR-590-5p promoter region into the pMIR-REPORT Luciferase plasmid. (d) HEK-293T cells
were transfected with a renilla luciferase plasmid plus a luciferase plasmid containing WT-PPAR-𝛾 or mut-PPAR-𝛾 promoter region; then thecells were stimulated by Rosiglitazone (PBS as negative control) for 24 h. Luciferase activities were determined by the ratio of pMIR-REPORT
plasmid to pCMV-Renilla control (n=3, ∗ P<0.05).

The promoter region (-410∼-390 bp) of miR-590-5p wasdetermined by PCR in the anti-PPAR-𝛾 immunoprecipitatedDNA. The recruitment of PPAR-𝛾 to this region was only
observed in PPAR-𝛾 transfected but not in PPAR-𝛾RNLS
transfected cells (Figure 4(b)). Next, the PPAR-𝛾 binding
motif was mutated and cloned into the pMIR-REPORT
Luciferase plasmid, both WT- and mut-type of PPAR-𝛾
promoters were transfected into HEK-293T cells, respectively(Figure 4(c)), and cells were stimulated by Rosiglitazone at
48 h after transfection. The result of luciferase expression
activity suggested that gene transcription could be driven by
the nuclear recruitment of PPAR-𝛾 to this region of the miR590-5p promoter (Figure 4(d)).

4. Discussion
This study demonstrates that Rosiglitazone treatment effectively protects the high-fat diet-induced ApoE−/− micethrough reducing the occurrence and development of
atherosclerosis. During the process of atherosclerosis, LOX1 upregulation plays a crucial role in the uptake of oxLDL in endothelium of the arterial wall. Here, we revealthat Rosiglitazone-mediated PPAR-𝛾 activation suppresses

the expression of LOX-1 in ox-LDL stimulated HUVECs via
activating the transcription of miR-590-5p.
Rosiglitazone belongs to the family of Thiazolidinedione(TZD), which is widely used in the treatment of type 2
diabetes, inflammation, and atherosclerosis. Emerging evidence indicated that LOX-1 could be negatively regulated by
PPAR-𝛾 activation in the treatment of atherosclerosis withTZDs [9, 22]. LOX-1 mediated suppression of ABCA1 inmacrophages could be reversed by TZDs, which improved
the reverse cholesterol transport and prevented the foamcell formation and ox-LDL accumulation on the arterial wallin the development of atherosclerosis [23, 24]. However,the precise role of PPAR-𝛾 in ox-LDL-induced endothelial
cells remained unclear. We previously revealed that Ciglitazone treatment also suppressed LOX-1 in ox-LDL-induced
endothelial cells and improved angiogenesis by activating
PPAR-𝛾 [25]. In this study, we further confirmed that PPAR-𝛾
activation could inhibit the upregulation of LOX-1 and LOX-1
mediated oxidative stress and adhesion of inflammatory cellsby targeting miR-590-5p.
In the present study, we found that miR-590-5p was
significantly downregulated in ox-LDL-stimulated HUVECs
in a dose-dependent manner. Overexpression of miR-5905p significantly reversed LOX-1 expression and repressed8LOX-1 mediated protein levels of ICAM-1, VCAM-1, andp47phox in ox-LDL-induced HUVECs. The inhibitory role ofmiR-590-5p on LOX-1 signaling was observed in both oxLDL-induced and AngII-induced HUVECs [11, 26]. miR590-5p mimics attenuated cell apoptosis, reduced ROS production, and improved angiogenesis through targeting the
3’-UTR of LOX-1 gene [11]. Consistent with these results,
we further revealed the upstream regulator of miR-590-5p.
The activity of microRNAs could be extensively regulatedby the nuclear transcription factors. PPAR-𝛾, also named
NR1C3 (nuclear receptor subfamily 1, group C, member 3),
is regarded as one kind of transcription factors encodedby the PPARG gene. In the proximal promoter region of
miR-590-5p, we observed one genetically conserved bindingsite for PPAR-𝛾 [27, 28]. This conserved core sequence
failed to be detected by ChIP-PCR assay in PPAR-𝛾RNLS
transfected HUVECs after stimulating with Rosiglitazone,
suggesting that PPAR-𝛾 nuclear translocation is requiredfor its binding with the DNA promoter of miR-590-5p. In
the previous studies, Rosiglitazone treatment increased thenuclear expression of PPAR-𝛾 in bone marrow stem cell
(BMSC) and ECs, which promoted cell differentiation andadipogenesis [29, 30]. We also reported the event of PPAR𝛾 nuclear translocation in Ciglitazone-pretreated dendritic
cells [31]. Nuclear translocated PPAR-𝛾 can be either anactivator or a repressor of gene transcription. For example,
PPAR-𝛾 is required to mediate the antioxidant effect andpromotes the production of antioxidant properties in theendothelium by forming a transcriptional complex withretinol binding protein 7 (RBP7) [32]. PPAR-𝛾 activation by
Rosiglitazone also blocks NFATc1 from binding to its own
promoter [33]. In this study, we did not test the possibility
whether nuclear translocation of PPAR-𝛾 promoted thetranscription of miR-590-5p in coordination with the othertranscriptional factors. But Rosiglitazone induced miR-5905p promoter activity significantly attenuated in HUVECs
after mutating the DNA binding site for PPAR-𝛾, indicating
a function role of nuclear PPAR-𝛾 in regulating miR-590-5ptranscription.
Previous studies showed a critical role of PPAR-𝛾 inpreventing insulin resistance and dysregulation of lipid
metabolism through regulating microRNA transcription[34–36]. Multiple microRNAs, such as miR-125a, miR-424,
and miR-503, that are transcriptionally activated by PPAR𝛾 are also found in inflammation-mediated angiogenesis inendothelial cells [15, 16]. Here, we observed that miR-590-5p
could be regulated by PPAR-𝛾 in HUVECs in a Rosiglitazonedependent manner, which effectively suppressed LOX-1 andLOX-1 mediated inflammation and oxidative stress. Ourfindings raise the possibility that one kind of TZD drugs
commonly used in diabetic therapy also has tremendous therapeutic potential in treatment of endothelial injury related
atherosclerosis.

Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request.

PPAR Research

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Authors’ Contributions
Lei Xu, Gang Zhao, and Hong Zhu contributed equally to thiswork.

Acknowledgments
This study was funded by grants from the National Natural Science Foundation of China (81700377, 81770274, and81300097) and Natural Science Foundation of Science andTechnology Commission of Shanghai (13ZR1406800 and
16ZR1406200).

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