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Data on myeloperoxidase-oxidized low-density lipoproteins stimulation of cells to
induce release of resolvin-D1
Dufour, Damien; Khalil, Alia; Nuyens, Vincent; Rousseau, Alexandre; Delporte, Cédric;
Noyon, Caroline; Cortese, Melissa; Reyé, Florence; Pireaux, Valérie; Nève, Jean;
Vanhamme, Luc; Robaye, Bernard; Lelubre, Christophe; Desmet, Jean Marc; Raes, Martine;
Boudjeltia, Karim Zouaoui; Van Antwerpen, Pierre
Published in:
Data in Brief
DOI:
10.1016/j.dib.2018.03.131
Publication date:
2018
Document Version
Publisher's PDF, also known as Version of record
Link to publication
Citation for pulished version (HARVARD):
Dufour, D, Khalil, A, Nuyens, V, Rousseau, A, Delporte, C, Noyon, C, Cortese, M, Reyé, F, Pireaux, V, Nève, J,
Vanhamme, L, Robaye, B, Lelubre, C, Desmet, JM, Raes, M, Boudjeltia, KZ & Van Antwerpen, P 2018, 'Data on
myeloperoxidase-oxidized low-density lipoproteins stimulation of cells to induce release of resolvin-D1', Data in
Brief, vol. 18, pp. 1160-1171. https://doi.org/10.1016/j.dib.2018.03.131
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Data article
Data on myeloperoxidase-oxidized low-density
lipoproteins stimulation of cells to induce release
of resolvin-D1
Damien Dufour
a,n, Alia Khalil
b, Vincent Nuyens
b,
Alexandre Rousseau
b, Cédric Delporte
a, Caroline Noyon
a,
Melissa Cortese
a, Florence Reyé
a, Valérie Pireaux
d, Jean Nève
a,
Luc Vanhamme
c, Bernard Robaye
f, Christophe Lelubre
b,
Jean-Marc Desmet
e, Martine Raes
d,
Karim Zouaoui Boudjeltia
b, Pierre Van Antwerpen
aa
Laboratory of Pharmaceutical Chemistry and Analytical Platform of the Faculty of Pharmacy, Faculty of Pharmacy, Université Libre de Bruxelles, Brussels, Belgium
bLaboratory of Experimental Medicine (ULB 222 Unit), CHU de Charleroi, A. Vésale Hospital, Université Libre de Bruxelles, Montigny-le-Tilleul, Belgium
c
Laboratory of Neurovascular Signaling, Universite libre de Bruxelles, Gosselies, Belgium d
URBC-Narilis, University of Namur, 5000 Namur, Belgium e
Unit of Dialysis, CHU de Charleroi, A. Vésale Hospital, Université Libre de Bruxelles, Montigny-le-Tilleul, Belgium
f
Institute of Interdisciplinary Research, IRIBHM, Universite Libre de Bruxelles, Gosselies, Belgium
a r t i c l e i n f o
Article history: Received 10 March 2018 Accepted 28 March 2018 Available online 4 April 2018
a b s t r a c t
This article present data related to the publication entitled“Native and myeloperoxidase-oxidized low-density lipoproteins act in synergy to induce release of resolvin-D1 from endothelial cells” (Dufour et al., 2018). The supporting materials include results obtained by Mox-LDLs stimulated macrophages and investigation performed on scavenger receptors. Linear regressions (RvD1 vs age of mice and RvD1 vs CL-Tyr/Tyr) and Data related to validation
Contents lists available at
ScienceDirect
journal homepage:
www.elsevier.com/locate/dib
Data in Brief
https://doi.org/10.1016/j.dib.2018.03.131
2352-3409/& 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
DOI of original article:https://doi.org/10.1016/j.atherosclerosis.2018.03.012
nCorresponding author. Phone+32 2650.52.49, Fax: +32 2650 53.31. E-mail address:damien.dufour@ulb.ac.be(D. Dufour).
were also presented. The interpretation of these data and further extensive insights can be found in Dufour et al. (2018) [1].
& 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Speci
fications table
Subject area
Biochemistry
More speci
fic subject area
Resolvin D1 in atherosclerosis
Type of data
Table, text
file, graph, figure
How data was acquired
Mass spectrometry (LC-MS-/-MS system from Agilent Technologies
(Santa Clara, CA, USA): an Agilent 1290 In
finity Binary - UHPLC
system coupled to a mass spectrometer Agilent Jet Stream
electro-spray ionization source (AJS)-Triple Quadrupole (QQQ) 6490 series)
Rt-PCR
Data format
Analyzed
Experimental factors
Samples were treated by liquid-liquid extraction before analysis
Experimental features
RvD1 and precursors (17S-HDHA and DHA) were quanti
fied in cell
supernatant or in plasma.
Data source location
Brussels, Belgium
Data accessibility
The data are provided with this article
Value of the data
Validation of method was an important part of this work and we showed how it was developed.
Data show how scavenger receptors, usually involved in oxidized LDLs recognition, were analyzed
and how they could be involved in RvD1 synthesis.
Correlation was established between level of RvD1 and Cl-Tyr/Tyr ratio from healthy donors and
between level of RvD1 and age in mice. These results are illustrated here.
Because HMEC have shown ability to produce RvD1, we assay the production of RvD1 by
mono-cytes (THP-1).
1. Data
Different aspects of method validation for RvD1 and precursors analysis were described. Moreover,
we investigated many aspects of RvD1 mechanistic of production linked to Mox-LDLs stimulation of
endothelial cells and THP-1 macrophages.
1.1. Macrophages are apparently not essential for RvD1 production
It is generally accepted that EC and monocytes/macrophages are both required to produce RvD1.
However, it was shown that EC are able to produce RvD1 alone
[1]
. Therefore, we tested whether
Mox-LDLs induced RvD1 production in the presence of THP-1. Cells were incubated with 100
μ
g/ml of
Mox-LDLs and data showed an increased concentration of RvD1 (76
7 21 pg/ml) compared with
THP-1 cells incubated with 100
μ
g/ml non-physiologic Ox-LDLs (RvD1 35
7 16 pg/ml) or 100
μ
g/ml
LDLs-nat (RvD1 14
7 10 pg/ml). However, the differences were not statistically significant. An
increase in 17S-HDHA was also observed in the same conditions with a concentration of 439
7 32 pg/
ml when incubated with 100
μ
g/ml of Mox-LDLs and concentrations of 192
7 84 pg/ml and 202 7
25 pg/ml when incubated with Ox-LDLs or LDLs-nat, respectively. These data were again not
statis-tically signi
ficant (see
Fig. 1
).
2. Experimental design, materials and methods
2.1. Macrophages are apparently not essential for RvD1 production
Because we suspected that THP-1 may participate in RvD1 production, we also tested the effects of
Mox-LDLs, non-physiologic Ox-LDLs and LDLs-nat on the synthesis of RvD1 and 17S-HDHA by THP-1.
Ox-LDL S
uppl
.
Mox-LD
L Suppl
.
LDL-nat
S
uppl
.
LP
S
S
uppl
.
C
ont
rol
S
uppl
.
Ox
-L
D
L
Mox-LD
L
LDL-nat
Co
ntr
ol
0
1
2
3
Re
la
ti
v
e
r
e
s
p
ons
e
Ox-LDL S
uppl
.
Mox-LD
L Suppl
.
LDL-nat
S
uppl
.
LP
S
S
upp
l.
C
ont
rol
S
uppl
.
O
x-LDL
Mox-LD
L
LDL-nat
C
ont
rol
0.0
0.5
1.0
1.5
2.0
Re
la
ti
v
e
r
e
s
p
ons
e
DHA
Ox-LDL S
uppl
.
Mox-LD
L Suppl
.
LDL-nat
S
uppl
.
LP
S
S
uppl
.
C
ont
rol
S
uppl
.
Ox
-L
D
L
Mo
x-LD
L
LDL-nat
C
ont
rol
0.0
0.5
1.0
1.5
Re
la
ti
v
e
r
e
s
p
ons
e
Fig. 1. (A), (B), (C): Incubation of THP-1: Data for RvD1 (A), 17S-HDHA (B) and DHA (C) after 24 h of incubation of THP-1 with Ox-LDLs, Mox-LDLs or LDLs-nat with (light grey bars) or without (dark grey bars) supplementation of 10 ng/ml 17S-HDHA and DHA. Data are expressed as mean7 SD of four independent experiments compared to the average of four controls (control RvD1: 0.027 0.01 ng/ml; 17S-HDHA: 0.26 7 0.05 ng/ml; DHA: 21 7 2 ng/ml).
Fig. 2. (A), (B), (C): Chromatograms obtained by LC/MS-MS analysis of RvD1 from plasma of healthy donor without supple-mentation (A) or spiked by 0.5 ng/ml of standard (B) and compared to Internal standard RvD1-d5 (C).
Pl
asm
a Co
nt
ro
l
Pl
asm
a sp
ik
ed
Pla
sm
a
sp
ik
ed
+ LD
L
25
0 µg/m
l
Pl
asm
a s
pi
ke
d + LD
L
50
0 µg/m
l
0
5
10
15
20
R
e
la
ti
ve r
esp
o
n
se
Pl
asm
a Co
ntr
ol
Pl
asm
a s
pi
ke
d
Pl
asm
a
sp
ik
ed
+
L
D
L
250
µg
/m
l
Pl
asm
a
sp
ik
ed
+
L
D
L
500
µg
/m
l
0
2
4
6
8
10
***
*
R
e
la
ti
ve r
esp
o
n
se
DHA
Pl
asm
a Co
nt
ro
l
Pl
asm
a sp
ik
ed
Pl
asm
a
sp
ik
ed
+
L
D
L
250
µg
/ml
Pl
asm
a
sp
ik
ed
+
L
D
L
500
µg
/m
l
0.0
0.5
1.0
1.5
2.0
***
*
**
R
e
la
ti
ve r
esp
o
n
se
Fig. 3. (A), (B), (C): Effect of lipeamia on quantification of RvD1, 17S-HDHA and DHA: Quantification of RvD1 (A), 17S-HDHA (B) and DHA (C) in sample of plasma from healthy donor, spiked by RvD1, 17S-HDHA and DHA (0.5 ng/ml, 5 ng/ml and 100 ng/ ml respectively) and by different concentration of LDLs-nat (0, 250, 500µg/ml). Data are expressed as mean 7 SD of four independent experiments compared to the average of four controls obtained with by plasma not spiked (control RvD1: 0.037 0.01 ng/ml; 17S-HDHA: 0.627 0.04 ng/ml; DHA: 566 7 48 ng/ml). * p o 0.05; ** p o 0.01 and *** p o 0.001 by Tukey Test.
THP-1 were incubated with 200
μ
g/ml of Mox-LDLs, LDLs-nat or non-physiologic Ox-LDLs. The effect
of supplementation with 10 ng/ml of DHA and 17S-HDHA was also tested. Samples were puri
fied by
the previous method and RvD1, 17S-HDHA and DHA were quanti
fied using LC-MS/MS as illustrated in
Fig. 1
(
Figs. 2
–
12
). The quanti
fication of RvD1 and precursors was tested in the presence of different
LDL concentrations (see
Fig 2
). For details on the method validation, see Dufour et al. (2018) and
tables 1
and
2
. This method was used for quanti
fication of RvD1 and precursors in different matrices
like cells supernatant and after genesilencing or plasma (see
Fig 3
–
12
). See Dufour et al. (2018)
[1]
for
more information.
RvD1
Control
Mo
x
Angio
II
M
ox +
A
ngio II
Angio
II +
M
P
O
M
ox +
A
ngio II
+ MPO
H
OC
l
Mo
x +
HO
Cl
0.0
0.1
0.2
0.3
ng/
m
L
17S-HDHA
Control
Mo
x
Angio
II
M
ox +
A
ngio II
Angio
II +
M
P
O
Mo
x
+
A
ngio II
+ MPO
H
OC
l
Mo
x +
HO
Cl
0
1
2
3
4
5
ng/
m
L
DHA
Co
nt
ro
l
Mox
An
gi
o I
I
Mox
+ Ang
io II
A
ngi
o II
+
M
P
O
Mox
+ A
ng
io II +
M
P
O
HO
Cl
Mo
x +
HO
Cl
0
50
100
150
200
ng/
m
L
Fig. 4. (A), (B), (C): RvD1 by MPO system stimulation of HMEC: concentrations (ng/ml) obtained for RvD1 (A), 17S-HDHA (B) and DHA (C) after stimulation of HMEC by MPO system, Angiotensine II, Mox-LDLs or HOCl.
Co
nt
ro
l
Mox-LD
L 200 µ
g/
m
l
H
D
L 1000 µ
g/
m
l
LDL 1
000 µg/ml
Mox-LD
L + LD
L
Mox-LD
L +
H
D
L
Mox
-LD
L + LD
L
+ HDL
0
5
10
15
N.D. N.D.ng/
m
L
Co
nt
ro
l
Mox-LD
L 200 µ
g/
m
l
H
D
L 1000
µg
/m
l
LDL 1
000 µg/ml
Mox
-LD
L + LD
L
Mox-LD
L + H
D
L
Mox-LD
L +
LD
L +
H
D
L
0
50
100
150
200
ng/
m
L
DHA
Co
nt
ro
l
Mox-LD
L 200 µ
g/
m
l
H
D
L 1000
µ
g/
m
l
LDL 1
000 µg/ml
Mox-LD
L + LD
L
Mox
-LD
L +
HD
L
Mox-LD
L + LD
L
+ HDL
0
100
200
300
400
ng/
m
L
Fig. 5. (A), (B), (C): Effect of LDLs-nat, HDLs and Mox-LDLs on HMEC: Quantification of RvD1 (A), 17S-HDHA (B) and DHA (C) in supernatants of cells stimulated by LDLs-nat (1000µg/ml), HDLs (1000 µg/ml) and Mox-LDLs (200 µg/ml) for 24 h at 37 °C. Data are expressed as mean7 SD of four independent experiments. Control was carried out on by HMEC alone.
Fig. 6. Validation of LDLR gene silencing efficiency using siRNA: RNA was extracted and analyzed by qRT-PCR. Expression levels were normalized to GAPDH. Values are expressed as fold change compared to the control. Data were evaluated by One-Way ANOVA test
RvD1
Ctr Sc SiRN A Mox -LDLs Mox -LDLs + Si RNA SCAR B1 Mox -LDLs + SiRNA LDLR Mox -LDLs + SiRNA LOX1 Mox -LDLs + SiRNA Pool Ox-LDLs Ox-LDLs + Si RNA SCAR B1 Ox -LDLs + SiRNA LDLR Ox-LDLs + Si RNA LOX1 Ox -LDLs + Si RNA Pool 0 5 10 15R
e
la
ti
ve r
esponse
17S-HDHA
Ct
r
Sc
Si
RNA
Mo
x-
LDL
s
Mo
x-
LDL
s + Si
RN
A
SC
A
RB1
Mo
x-
LD
Ls
+ Si
RN
A LDL
R
Mo
x-
LDL
s + Si
RN
A LOX
1
Mo
x-
LDL
s +
SiRN
A Poo
l
O
x-LD
Ls
Ox
-L
DLs
+ Si
RNA
S
C
AR
B1
O
x-LD
Ls
+ Si
RNA
L
D
LR
Ox
-L
DLs
+ Si
RNA
L
O
X1
Ox
-LD
Ls
+
S
iR
N
A
Po
ol
0
1
2
3
4
R
e
la
ti
ve r
esp
o
n
se
DHA
Ct
r
Sc
Si
RNA
Mo
x-
LDL
s
Mo
x-
LDL
s + Si
RN
A SCA
RB1
Mo
x-
LDL
s + Si
RN
A LDL
R
Mo
x-
LDL
s + Si
RN
A LOX
1
Mo
x-
LDL
s + Si
RN
A Poo
l
Ox
-L
D
Ls
Ox
-L
DLs
+ Si
RNA
S
C
AR
B1
Ox
-L
DLs
+ Si
RNA
LD
LR
Ox
-L
DLs
+ Si
RNA
L
O
X1
Ox
-L
DLs
+ Si
RNA
P
oo
l
0
1
2
3
R e la ti v e re s pons eFig. 7. (A), (B), (C): Control of integration of Mox-LDL or Ox-LDL by LDL receptor: Relative response of RvD1 (A), 17S-HDHA (B) and DHA (C) calculated after incubation of endothelial cells with SiRNA LDLr, SiRNA SCARB1, SiRNA LOX-1 or all SiRNA pooled in presence of Mox-LDL or Ox-LDL (100µg/ml). Controls were realized by endothelial cells without transection or transfected by scrambled siRNA.
2.2. in vivo macrophage subpopulations analysis using
flow cytometry
100
µL of total blood were incubated for 15 min at RT with PE mouse anti-human CD14 and V500
mouse anti-human CD16 antibodies (Becton Dickinson, Franklin Lakes, NJ, USA) as well as with mouse
anti-human CD86-FITC and anti-human CCR2-APC monoclonal antibodies (Miltenyi Biotec, Bergisch
Gladbach, Germany) for determining the M1 polarization or with mouse anti-human CD206-FITC,
anti-human CXCR3-APC and anti-human CD163-VioBlue monoclonal antibodies (Miltenyi Biotec,
Bergisch Gladbach, Germany) for the M2 polarization. Red blood cells were then eliminated by adding
BD FACS Lysing Solution (dilution: 1/20) (Becton Dickinson, Franklin Lakes, NJ, USA) to total blood and
remaining cells were washed twice with 1 mL of running buffer. Cells were
finally resuspended in 300
µL of running buffer for analysis. The matching isotype controls were used for each antibody in order
to de
fine the threshold. The analysis was performed using the MACSQuant Analyzer 10 (Miltenyi
Biotec, Bergisch Gladbach, Germany), applying a gating strategy based on the SSC vs PE gate (CD14),
selecting the monocyte population. Classical monocytes were de
fined based on a high expression of
Fig. 8. (A), (B), (C): Validation of gene silencing efficiency using siRNA: HMEC-1 cells were transfected with scrambled siRNA (sc siRNA) or LDLR siRNA for 48 h then treated with Mox-LDL or OxLDL (100µg/ml) for another 24 h. RNa was then extracted and analyzed by qRT-PCR using primers specific for LDLR. The expression level was normalized to GAPDH. Values are expressed as the fold change to the control. Data were evaluated by one-way ANOVA test: * po 0.05, ** p o 0.01 and *** p o 0.001 vs control were considered significant.
RvD1
Ctr Mo x-LDL s 24 h Mo x-LDL s + Si RN A 24h Mo x-LDL s 48 h Mo x-LDL s + Si RN A 48 h O x-LDLs 24 h Ox-L DLs + Si RNA 2 4h Ox-LDLs 48h Ox-LDLs + Si RNA 48h 0 2 4 6 8 10R
e
la
ti
v
e
r
e
s
pons
e
17S-HDHA
Ctr
Mo
x-LDLs 24h
Mo
x-LDLs +
SiRNA
24h
Mo
x-
LD
Ls 48h
Mo
x-LDLs +
SiRNA
48h
O
x-LDLs 24h
Ox-LD
Ls +
SiRNA 24h
O
x-LDLs 48h
Ox-LDLs
+ SiRNA 48h
0.0
0.5
1.0
1.5
R e la ti ve r e s p o n s eDHA
Ctr
Mo
x-LDLs 24h
Mo
x-LDLs +
SiRNA
24h
Mo
x-LDLs 48h
Mo
x-LDLs +
SiRNA
48h
O
x-LDLs 24h
Ox-LDLs +
SiRNA
24h
O
x-LDLs 48h
Ox-LDLs +
SiRNA
48h
0.0
0.5
1.0
1.5
R
e
la
ti
ve r
esp
o
n
s
e
Fig. 9. (A), (B), (C): Effect of time on SiRNA LOX-1: Relative intensity of RvD1 (A), 17S-HDHA (B) and DHA (C) calculated by normalization to control after incubation of endothelial cells with SiRNA LOX-1 in presence of Mox-LDL or Ox-LDL (100µg/ml) for 24 h or 48 h. Controls were realized by endothelial cells without transection.
CD14 and a low expression of CD16 (CD14
þCD16-). Intermediate monocytes were defined based on a
high expression of CD14 and CD16 (CD14
þCD16þ), while non-classical monocytes were
character-ized by a low expression of CD14 and a high expression of CD16 (CD14-CD16
þ) (
Tables 1
–
3
).
Linear reg. RvD1 vs age of mice
0 200 400 600 800 0.00 0.05 0.10 0.15 RvD1 days ng /m L
Fig. 11. Linear regression of RvD1 versus age of mice s (n¼ 65, ♀ ¼ 40, ♂¼ 25, middle age ¼ 291 7 270 days, R2¼ 0.08782, p¼ 0.0165, slope ¼ −0.00006673 to −0.000006936).
linear reg. RvD1 vs Cl-Tyr/Tyr
0.00 0.02 0.04 0.06 0.08 0 1e-005 2e-005 3e-005
RvD1
Cl-Tyr/Tyr
Fig. 12. Linear regression of RvD1 versus ratio Chloro-tyrosine/tyrosine from healthy donors (n¼ 23, R2¼ 0.1809, p ¼ 0.0430, slope¼ −0.0001060 7 0.00004921).
***
***
Fig. 10. Quantification of RvD1 after 24 h of incubation of endothelial cells with DMSO (vehicle), DPI (NADHP oxidase and NOS inhibitor), ML351 (lipoxygenase inhibitor), MAFPF (PLA2inhibitor), dexamethasone (anti-inflammatory), water (vehicle) Trolox (antioxidant) in presence of Mox-LDL compared to a control. Data are given as relative response normalized of 4 independent experiments. Data were considered as significant (MAFP vs DMSO and Trolox vs Water) by ANOVA: p o 0.001 (***).
Transparency document. Supporting information
Transparency data associated with this article can be found in the online version at
http://dx.doi.
org/10.1016/j.dib.2018.03.131
.
Reference
[1] D. Dufour, A. Khalil, V. Nuyens, et al., Native and myeloperoxidase-oxidized low-density lipoproteins act in synergy to induce release of resolvin-D1 from endothelial cells. Atherosclerosis. 272, 2018, 108-117.http://dx.doi.org/10.1016/j.atherosclerosis. 2018.03.012.
Table 1
Transitions optimization: Transitions associated to collision energy used like quantifier or qualifier and retention time in minutes for each compound. These transitions were obtained using Optimizer program from Agilent Technologies and were confirmed by manual optimization.
Compounds (M-H)- Quantifier m/z (coll. Energy) Qualifier m/z (coll. Energy) Retention time (min) RvD1 375.22 141.1 (16 eV) 215.1 (16 eV) 4.8 RvD1-d5 380.25 141.1 (13 eV) 220 (17 eV) 4.8
17S-HDHA 343.23 281.3 (8 eV) 325.3 (8 eV) 7.0
PGE2-d4 355.2 319.1 (4 eV) 275.2 (12 eV) 4.5
DHA 327.23 283.2 (8 eV) 58.9 (40 eV) 8.2
DHA-d5 332.26 288.0 (5 eV) 234.3 (9 eV) 8.2
Table 2
Data of validation: Recovery and coefficients of variation (CV) obtained using liquid/liquid purification of plasma spiked with two different concentrations of RvD1 (1 ng/ml and 0.5 ng/ml), 17S-HDHA (10 ng/ml and 5 ng/ml) and DHA (200 ng/ml and 100 ng/ml).
Product Concentration (ng/ml) Recovery (%) CV (%)
RvD1 1 86.17 8.1 9.4 0.5 90.27 3.1 3.4 17S-HDHA 10 45.27 12.0 26.5 5 53.07 7.7 14.6 DHA 200 100.17 13.2 13.2 100 104.57 5.4 5.2 Table 3
List of primers: primers used for qRT-PCR.
Gene Reverse sequence (5’-3’) Forward sequence (5’-3’)
OLR1 CGTGACTGCTTCACTCTCTCAT TCAGACACCTGGGATAATTGCAT
LDLr CCCTGACGAATTCCAGTGCT GAGTGTCACATTAACGCAGCC
SCARB1 GGCCATTCAGGCCTATTCTGA TCCTCAGGACCCTACAGTTTTG
