Am J Physiol Heart Circ Physiol 309: H1894 –H1903, 2015. First published October 9, 2015; doi:10.1152/ajpheart.00289.2015.
Soluble epoxide hydrolase is involved in the development of atherosclerosis and arterial neointima formation by regulating smooth muscle cell migration Qingjie Wang,1,2* Leijun Huo,1* Jinlong He,3 Wenshuang Ding,1 Hang Su,1 Dongping Tian,1 Carrie Welch,4 Bruce D. Hammock,5 Ding Ai,3 and Yi Zhu3 1
Department of Pathology, Shantou University Medical College, Shantou, Guangdong, China; 2Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai, China; 3Department of Physiology and Pathophysiology, Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Medical University, Tianjin, China; 4Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York; and 5 Department of Entomology and Comprehensive Cancer Center, University of California, Davis, California Submitted 22 April 2015; accepted in final form 18 August 2015
Wang Q, Huo L, He J, Ding W, Su H, Tian D, Welch C, Hammock BD, Ai D, Zhu Y. Soluble epoxide hydrolase is involved in the development of atherosclerosis and arterial neointima formation by regulating smooth muscle cell migration. Am J Physiol Heart Circ Physiol 309: H1894 –H1903, 2015. First published October 9, 2015; doi:10.1152/ajpheart.00289.2015.—Epoxyeicosatrienoic acids (EETs) have beneficial effects on cardiovascular disease. Soluble epoxide hydrolase (sEH) metabolizes EETs to less active diols, thus diminishing their biological activity. sEH inhibitors can suppress the progression of atherosclerotic lesions in animal models. However, the regulation of sEH in vascular smooth muscle cells (VSMCs) and role of sEH in patients with atherosclerosis have not been evaluated. We hypothesize that sEH in VSMCs plays a pivotal role in atherosclerosis and injury-induced neointima formation. In this study, sEH expression in human autopsy atherosclerotic plaque was determined by immunohistochemistry. In cultured rat and human VSMCs, the phenotypic switching marker and sEH expression induced by plateletderived growth factor-BB (PDGF-BB) were examined by Western blot analysis. Carotid-artery balloon injury was performed after adenovirus-mediated overexpression of sEH or oral administration of a potent sEH inhibitor in Sprague-Dawley rats. sEH was highly expressed in VSMCs of the intima and media within human atherosclerotic plaque. In vitro, PDGF-BB upregulated the expression in VSMCs after transcription and promoted cell proliferation and migration; the latter effect could be largely attenuated by an sEH inhibitor. Adenovirus-mediated overexpression of sEH could mimic the effect of PDGF-BB and induce VSMC proliferation and migration. In vivo, the sEH inhibitor led to a significant decrease in injury-induced neointima formation in a rat carotid-artery injury model. These data establish the effect of sEH expression on atherosclerotic progression and vascular remodeling after injury, thus identifying a novel integrative role for sEH in VSMC phenotypic modulation and migration. Blocking sEH activity may be a potential therapeutic approach for ameliorating vascular occlusive disease. soluble epoxide hydrolase inhibitor; atherosclerosis; vascular smooth muscle cell; migration; neointima formation
NEW & NOTEWORTHY
In this study, we found that soluble epoxide hydrolase (sEH) was highly expressed in vascular smooth muscle cells (VSMCs) of human atherosclerotic plaque and in rat neointima. Inhibition of sEH largely attenuated platelet-derived growth factor* Q. Wang and L. Huo contributed equally to this work. Address for reprint requests and other correspondence: Y. Zhu, Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Medical Univ., Tianjin, 300070, China (e-mail: [email protected]). H1894
BB-induced VSMC migration in vitro and injury-induced neointima formation in vivo, which suggests that sEH inhibition may be a potential therapeutic target for cardiovascular diseases. EPOXYEICOSATRIENOIC ACIDS (EETs), derived from arachidonic acid, have several beneficial effects on cardiovascular homeostasis, including the ability to hyperpolarize vascular smooth muscle cells (VSMCs), dilate coronary arteries, and suppress adhesion molecules. Soluble epoxide hydrolase (sEH) metabolizes EETs to their corresponding dihydroxyeicosatrienoic acids, which limits the availability of EETs (16). Several models of atherosclerosis have suggested that inhibition of sEH, with an ensuing decrease in EET degradation, may attenuate atherosclerosis development and aneurysm formation (3, 14, 15, 19, 25). Treatment with sEH inhibitors also attenuated cardiohypertrophy in rats with angiotensin II treatment and in a murine model with transverse aortic constriction (21). However, the involvement of sEH in the development of human cardiovascular disease is unclear. VSMCs, a major blood vessel cell type, play major roles in maintaining vessel integrity and function. The migration of VSMCs from media to intima is pivotal for intimal thickening in cardiovascular disease. VSMCs in adult animals are plastic and retain their ability to switch rapidly from a contractile excitable phenotype to a migratory synthetic phenotype in response to injury (11). This modulation is the common fundamental biological mechanism of VSMC-related cardiovascular diseases such as atherosclerosis and neointimal formation after arterial injury. A characteristic of synthetic VSMCs is that they have low levels of differentiation marker proteins, including ␣-smooth muscle actin (␣-SMA) and smooth muscle protein 22␣ (SM22␣), which are required for the VSMC normal contractile function (12, 13). Given the important role of VSMCs in atherosclerosis and neointima formation after arterial injury and the effect of sEH/EETs on the cardiovascular system, we hypothesize that sEH in VSMCs plays a pivotal role in vascular remodeling. In the current study, we investigated the role of VSMC sEH in atherosclerotic and injury-induced neointima formation. We found that sEH was highly expressed in VSMCs of human atherosclerotic plaques. In vitro, sEH played a critical role in VSMC migration by inhibiting platelet-derived growth factor-BB (PDGF-BB)-induced differentiation. Pharmacological inhibition of sEH in a balloon-injured carotid artery model decreased neointima formation and injury-induced VSMCspecific gene expression. sH may be a potential therapeutic
0363-6135/15 Copyright © 2015 the American Physiological Society
http://www.ajpheart.org
H1895
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
target for attenuating VSMC dedifferentiation and migration under pathological conditions.
Table 1. Age and gender information of autopsy samples Age
MATERIALS AND METHODS
Reagents. Cycloheximide (CHX) and Hoechst stain reagent were from Sigma-Aldrich (St. Louis, MO). PDGF was from R&D Systems (Minneapolis, MN). Primary antibodies for sEH (sc-25797), osteopontin (OPN, sc-21742), GAPDH, CD68 (sc-9139), and polyperoxidase-anti-mouse/rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against smooth muscle ␣-actin (AA132) were from Beyotime Institute of Biotechnology (Beijing, China), and DyLight 488-labeled goat anti-rabbit IgG (H⫹L) and DyLight 594labeled goat anti-mouse IgG (H⫹L) were from Zhongshan Golden Bridge (Beijing, China). Human samples and analysis. Autopsy case records from 2001 to 2010 at the Verification Centre of Forensic Medicine affiliated with Shantou University were reviewed, from which 97 formalin-fixed
Sex
Group
Range
Mean
Male
Female
Nondiseased vessel, n ⫽ 36 Atherosclerotic plaques, n ⫽ 61
18–60 24–77
32.93 ⫾ 10.50 46.18 ⫾ 13.47
20 58
16 3
paraffin embedded coronary or coronary artery sections were chosen and stained with hematoxylin and eosin (H&E) to evaluate the degree of atherosclerosis. On the basis of H&E staining, sections were separated into two groups: one nondiseased and one with atherosclerotic plaque. Ponceau/Victoria blue (P/VB) was used to stain the elastic fibers surrounding vessels and proteins in cells, respectively. Sixty-one samples exhibited atherosclerotic plaques and 36 showed nonplaque arteries. The age and sex of individuals in each group are listed in Table 1. Analysis of sEH location and intensity was per-
H&E staining
A
Atherosclerotic plaques
Non-diseased vessels
B
f
C
P/VB
c
d
e
f
**
3
h
α-SMA
sEH b
4
CD68 d
g
a
Relative protein level of sEH
D
c
b
e
α-SMA
sEH
P/VB a
Fig. 1. Soluble epoxide hydrolase (EH) protein level is upregulated in atherosclerotic lesions of human coronary arteries. A: hematoxylin and eosin (H&E) staining of human coronary artery sections (nondiseased vessel, n ⫽ 36; atherosclerotic plaque, n ⫽ 61). B: representative immunohistochemical analysis of atherosclerotic patient coronary artery cross sections; a–d: immunostaining with Ponceau/ Victoria blue (P/VB) and antibodies for soluble EH (sEH), ␣-smooth muscle actin (␣-SMA), and CD68 (bar ⫽ 500 m); e–h: higher amplification of boxes (bar ⫽ 100 m). C: immunohistochemical staining with P/VB, sEH (arrows) and ␣-SMA. Top, bar ⫽ 100 m; bottom, higher magnification of boxes, bar ⫽ 50 m. D: quantification of sEH in intima and media. Nondiseased vessels, n ⫽ 36; atherosclerotic plaque, n ⫽ 61; data are means ⫾ SE, **P ⬍ 0.001, media of nondiseased vessel group vs. media of atherosclerotic plaque group. Bar ⫽ 100 m.
Non-diseased vessel group Atherosclerotic plaque group
2 1 0
Intima
Media
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
H1896
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
formed in a blinded fashion. The average density (integral optical density/area) was measured with Image-Pro Plus 6.0, and the result was determined as the sum of five different fields of each section. The level of sEH average density was normalized to that of nondiseased vessels in intima. The study was carried out in accordance with the Declaration of Helsinki and The Belmont Report (Washington, DC: U.S. Department of Health &Human Services, 1979), and was approved by the Ethics Committee of Shantou University Medical College. Animal artery injury. Male Sprague-Dawley rats (210 to 230 g) were housed in a temperature-controlled room with a 12-h:12-h light-dark cycle and had free access to standard chow and water. The sEH inhibitor 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) was designed and synthesized in Bruce Hammock’s laboratory at Columbia University in New York. When compared with early generations of competitive sEH inhibitors, TUPS exhibits high potency (IC50 ⫽ 7.0 nM in rat), excellent oral availabil-
A
ity, and pharmacokinetic properties (1). TUPS was dissolved and delivered at 0.65 mg·kg⫺1·day⫺1 in drinking water for 10 or 14 days as was previously reported (3), starting 7 days before surgery. A vehicle was administered to the control group. At 3 or 7 days after carotid artery injury, all animals (n ⫽ 7 to 9 per group) were anesthetized by CO2, and arteries were harvested. The protocols in the investigation followed those in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Publication No. 85-23, Revised). All animal experimental protocols were approved by the Shantou University Institutional Animal Care and Use Committee. Surgery was performed as described (20). Briefly, rats were anesthetized by injection of pentobarbital sodium (30 mg/kg ip). A balloon catheter of 1.5 mm in diameter (Medtronic, Minneapolis, MN) was introduced through the left external carotid artery and advanced 4 cm toward the thoracic aorta. The balloon was distended and pulled back to the bifurcation with constant rotation. This procedure was repeated two more times to ensure complete endothelial denudation. Contralat-
B
PDGF (24hr) 0
5
10
20
** 2
*
**
1
0
D
4
Human VSMC **
3 2
**
**
**
1 0 0
5
10
20
40 (ng/ml)
12h
24h
2.5 2.0
0.5
** **
**
**
** **
**
*
0.5 0
Human VSMC
2.5
*
2.0
*
1.5
**
**
1.0 0.5
** **
**
1.0
0 0
F
**
*
1.5
4
8
12
24 (h)
6
sEH-LUC activty
Relative mRNA level
Relative protein level of sEH Relative protein level of sEH
sEH relative protein level Relative protein level of sEH
3
1.0
0
8h
GAPDH
E 1.5
PDGF (ng/ml)
4h
sEH
GAPDH
C
0h
40 (ng/ml)
sEH
Fig. 2. Platelet-derived growth factor (PDGF) induces sEH expression in rat and human vascular smooth muscle cells (VSMCs). Rat VSMCs were treated with PDGF-BB for 24 h at the indicated dose (A) or 20 ng/ml PDGF for different times (B). Western blot analysis of sEH protein expression and quantification. *P ⬍ 0.05, **P ⬍ 0.01 compared with control. A paired t-test was performed. Human VSMCs were treated with PDGF-BB for 24 h at the indicated dose (C) or 20 ng/ml of PDGF for different times (D). Quantification of sEH protein expression is shown. *P ⬍ 0.05, **P ⬍ 0.01 compared with control. A paired t-test was performed. E: rat VSMCs were incubated with PDGF-BB at the indicated dose for 24 h. RT-PCR analysis of mRNA levels of sEH and VSMC differentiation markers was performed. Messenger RNA levels were normalized to those of GAPDH. *P ⬍ 0.05, **P ⬍ 0.01 compared with control. A t-test was performed. F: rat VSMCs were transfected with human sEH promoter-Luc plasmids (⫺1,000 and ⫺471 bp) and treated with 20 ng/ml of PDGF-BB for 24 h. -Gal plasmid was used as an internal control. Data are means ⫾ SD of 3 independent experiments.
PDGF (20ng/ml)
Ctrl PDGF 4
2
**** 0
0 5 10 20 40
0 5 10 20 40
sEH
-SMA
0 5 10 20 40 0 5 10 20 40
SM-22α
Calponin 1
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
sEH-1000
sEH-471
H1897
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
In vitro scratch assay and transwell migration assay. For scratchinduced wound healing assay, rat or human VSMCs were cultured until they were 90% confluent. The monolayers were scratched with use of a p200 pipette tip as described (10) and pretreated with TUPS (1 M) or 14,15-EET (100 nM) for 30 min and then incubated with 20 ng/ml of PDGF-BB or PBS for 24 h in serum-free medium. The farthest distance that cells migrated from the wound edge was measured (average of 5 independent microscope fields for each of 3 independent experiments). Analysis of VSMC invasion involved a modified Boyden chamber coated with an 8-m barrier of reconstituted basement-membrane protein similar in composition to that surrounding VSMCs in vivo (Millipore, Billerica, MA). Assays of sEH activity. EET and 14,15-dihydroxyeicosatrienoic acid (DHET) were measured as previously reported (26). Briefly, cell lysate samples were spiked with 1 l of internal standard mixture (5 ng for each internal standard). The Waters Oasis-HLB cartridges (1 ml volume) were used for solid-phase extraction. The elutions were further dried and redissolved in 30% acetonitrile. 14,15-EET and its metabolite of sEH, 14,15-DHET, were measured with ultra-highperformance liquid chromatography (UPLC; Waters, Milford, MA) and a 5500 QTRAP mass spectrometer (AB Sciex, Foster City, CA) equipped with a Turbo Ion Spray electrospray ionization source (liquid chromatography coupled with tandem mass spectrometry; LC-MS/MS) as described previously (26). The DHET-to-EET ratio was used to indicate sEH activity. Statistical analysis. Results were analyzed by two-tailed Student’s t-test or one-way or a two-way ANOVA or linear regression as indicated, by use of GraphPad Prism software. A Bonferroni test was used to test for significant post hoc differences revealed by ANOVA. A Pearson 2 test was used to compare sEH positivity in the intima and media of nondiseased and atherosclerotic tissue by use of SPSS v11.5 (SPSS Inc, Chicago, IL). P ⬍ 0.05 was considered statistically significant.
A
Ctrl CHX (h)
0
1
2
PDGF
4
8
4
5
0
1
2
4
8
sEH GAPDH
B
0.5
0.0 1
Ln[(sEH) t/(sEH) 0]
eral carotid arteries underwent a similar operation without injury and served as sham-operated controls. Morphometric analysis of rat carotid arteries. Rat arteries were perfusion-fixed with 4% paraformaldehyde. Briefly, cryosections (6 m thick, 350 m apart) were taken from the middle portion of the balloon-injured segment, and eight sections of each sample were analyzed by H&E staining and with Spot Image software (Diagnostic Instruments, Australia). The intima and media areas and the circumference of the external elastic lamina were determined, and the ratio of intima to media area was calculated. VSMC culture. Rat VSMCs were isolated from thoracic aortas of male Sprague-Dawley rats (180 to 200 g) as described (9) and maintained in DMEM (Gibco) containing 20% fetal bovine serum (FBS) (Highclone). Rats were euthanized with an overdose of pentobarbital sodium (100 mg/kg ip) before the aorta was excised. Purity of VSMCs was verified by anti-␣-actin antibody staining, and cells between passages 2 to 4 were made quiescent by incubating in serum-free DMEM for 48 h at 37°C in a humidified atmosphere containing 5% CO2 for in vitro experiments unless stated otherwise. A human VSMC cell line (HA-VSMC) was purchased from FuDan IBS Cell Center and cultured in DMEM/F12 with 10% FBS (Gibco), 0.05 mg/ml vitamin C (Sigma), 0.03 g/ml EGF (PrimeGene Bio Tech, Shanghai, China), 0.01 mg/ml insulin (Sigma), 0.01 mg/ml transferrin (Sigma), and 10 ng/ml sodium selenite (Sigma). Adenovirus infection. The protocol of VSMCs infected with adenovirus sEH (Ad-sEH) or adenovirus-mediated green fluorescent protein (Ad-GFP) has been described elsewhere (3, 18, 23). The adenovirus was added to the medium and incubated for 24 h. The infection efficiency was detected by RT-PCR and Western blot analysis. Ad-GFP was used as a negative control. Quantitative RT-PCR. Total RNA was extracted by use of TRIzol (Invitrogen, Carlsbad, CA). RNA concentration and quality were determined by spectral absorption at 260 and 280 nm. Equal amounts of total RNA were reverse transcribed by use of the AMV Reverse Transcription System (Promega, Madison, WI). Quantitative RT-PCR of cDNA involved the Brilliant SYBR Green QPCR system, with GAPDH as an internal control. The rat nucleotide primer sequences were 5=-GCTCTCAAAAAGAAAGGATT-3= and 5=-TGACAGGACTCTATGAGGAA-3= for sEH, 5=-AGCATCCGACCTTG-3= and 5=-AGCATAGCCCTCATAG-3= for ␣-SMA, 5=-ACATGTTCCAGACTGTTGAC-3= and 5=-TGTCTGTGAACTCCCTCTTA-3= for SM22␣, 5=-GTCTGCTGAGGTAAAGAACA-3= and 5=-ACTCATTGACCTTCTTCACA-3= for Cnn1, and 5=-TCCCTCAAGATTGTCAGC-3= and 5=-AGCCCTTCCACGATGC-3 for GAPDH. Western blot analysis. VSMCs were washed with PBS and lysed in RIPA buffer supplemented with protease inhibitor cocktail (NovasyGen, China). Protein concentration was determined by the bicinchoninic acid protein assay kit (NovasyGen). Equal amounts of protein were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Germany), which were incubated with the primary antibodies for sEH, ␣-SMA, OPN, and GAPDH, then corresponding secondary antibodies; blots were developed by use of Super ECL Plus Detection Reagent (Applygen, China). Transient transfection and luciferase activity assay. Plasmids of the human sEH promoter, sEH-1000bp-Luc and sEH-471bp-Luc, were used for transient transfection. VSMCs at 80% confluence were transfected with 1 g of sEH promoter and 0.5 g of CMV-galactosidase by use of a Jet PEI (Polyplus, San Marcos, CA) for 24 h. Cells were treated with 20 ng/ml of PDGF-BB for another 24 h, and then lysed and collected for luciferase activity assay (Luciferase Reporter Assay System, Promega). Protein half-life measurements. VSMCs were pretreated with CHX (100 g/ml) for 30 min and then incubated with or without PDGF (20 ng/ml). Cells were harvested at 0, 1, 2, 4, and 8 h, and the amount of protein was detected by Western blot analysis. The kinetic constant and half-life of sEH protein with and without PDGF-BB were determined by linear regression analysis.
2
3
6
7
8
Time (h)
Time (h) -0.5
PDGF+CHX -1.0
K= -0.15± 0.01 t1/2 =4.60± 0.15 -1.5
-2.0
CHX K= -0.24± ± 0.01 t1/2 =2.47± 0.50
-2.5
Fig. 3. PDGF increases the stabilization of sEH protein in rat VSMCs. A: VSMCs were serum-starved for 48 h, then treated with cycloheximide (CHX) (100 g/ml) for the indicated time, with or without PDGF-BB (20 ng/ml). Western blot analysis of sEH protein level was normalized to GAPDH. Data are representative of 5 experiments. B: the slope of the decay line was calculated by standard linear regression analysis (P ⬍ 0.0001).
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
H1898
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
A
B
12h
Ctrl
4
PDGF
PDGF
Ctrl
**
PDGF+TUPS PDGF+EET
PDGF+EET
Relatived Migration Distance
3
PDGF+TUPS
** # * ##
** 2
**
##
** # 1
0 0
5
D
C Ctrl
10
15
Time (hours)
PDGF 40
PDGF+EET
Cell Number
30
PDGF+TUPS
**
20
** #
* ##
10 0
E
2.0
DHET/EET ratio (fold of control)
1.5
* #
1.0
0.5
0.0
Fig. 4. Migration of human VSMCs induced by PDGF was partly reversed by the sEH inhibitor 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoro-methoxyphenyl)-urea (TUPS) or epoxyeicosatrienoic acid (14,15-EET). A and B: confluent human VSMC monolayers were preincubated with TUPS (1 M) or 14,15-EET (100 nM) for 30 min and then treated with 20 ng/ml of PDGF. Scratch assay was performed. Images were taken at ⫻100 magnification. Data are means ⫾ SD of 5 experiments. *P ⬍ 0.05, **P ⬍ 0.01 compared with control; #P ⬍ 0.05 compared with PDGF alone. C: human VSMCs were seeded in the upper chamber of transwells. PDGF-BB (20 ng/ml) was added in the lower chamber as a chemoattractant with TUPS (1 M) or 14,15-EET (100 nM). Magnification is ⫻400. D: the mean cell number migrating across the filter was quantified in five randomly chosen fields. E: human VSMCs were incubated with or without TUPS (1 M) and treated with 20 ng/ml PDGF for 24 h, after which 14,15-EET and 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) were determined with LC/MS-MS and the DHET-to-EET ratio was calculated. Data are means ⫾ SD and analyzed by ANOVA from 5 independent experiments. *P ⬍ 0.05, **P ⬍ 0.01 compared with control; #P ⬍ 0.05, ##P ⬍ 0.05 compared with PDGF alone. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
H1899
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION RESULTS
of sEH in response to the VSMC migration inducer PDGF-BB. Rat and human primary VSMCs were treated with different doses of PDGF-BB for 24 h. sEH protein expression was induced by PDGF-BB at 10 ng/ml and peaked at 40 ng/ml (Fig. 2, A and C). In addition, PDGF treatment increased sEH protein levels at 8 and 4 h, in rat and human cells, respectively, which lasted for at least 24 h (Fig. 2, B and D). Thus PDGF induced sEH expression in both a dose- and time-dependent manner. The increase in sEH expression paralleled that of a reduced expression of contractile SMC markers such as ␣-SMA, calponin 1, and SM22␣. However, the mRNA levels of sEH and luciferase activity of the sEH promoter were not changed by PDGF-BB in VSMCs (Fig. 2, E and F). Therefore, PDGF-BB-induced regulation of sEH occurred posttranscriptionally. Because sEH was regulated by PDGF at the protein level, we next tested whether PDGF-BB enhanced the stability of sEH protein. We used CHX to block protein synthesis and measured the degradation of sEH in VSMCs. Soluble degradation was slower with PDGF-BB than control treatment (Fig. 3A). CHX treatment produced an sEH half-life of 2.5 ⫾ 0.5 min in control cells and 4.6 ⫾ 0.2 min in PDGF-treated VSMCs (Fig. 3B).
sEH protein is highly expressed in VSMCs in human atherosclerotic lesions. To investigate the role of sEH in atherosclerosis, we studied its expression in human coronary artery sections with polyclonal rabbit anti-human sEH antibody. Sections from 97 autopsy cases were divided into two groups: sections containing nondiseased vessels and sections containing atherosclerotic plaques (Fig. 1A) according to H&E staining of samples and pathology diagnosis. To further identify the sEH-positive cells, we immunostained human sections with P/VB and antibodies for sEH, ␣-SMA, and CD68. The expression of sEH corresponded to that of ␣-SMA rather than CD68 (Fig. 1B), which suggests that sEH was predominantly expressed in VSMCs in the intima and media (Fig. 1C). Moreover, the density of sEH was comparable in intima, which was significantly increased in the media of atherosclerotic plaques (Fig. 1D), so the high sEH expression in the VSMCs of atherosclerotic intima might be related to the development of atherosclerosis. PDGF-BB posttranscriptionally upregulated sEH expression in rat primary and human VSMCs. To explore a potential role for sEH in VSMC migration, we measured the expression
B
**
DHET/EET ratio (fold of control)
2.0 1.5 1.0 0.5 0.0
Ad-GFP
C
Ad-GFP
5
30
Ad-sEH
*
3
**
2 1 0
Ad-sEH
**
*
4
Cell Number
2.5
Relatived Migration Distance
A
0
10
20
10
0
20
Ad-sEH
Ad-GFP 7.5
2.5
5
7.5
sEH α-SMA OPN
E sEH protein level (relative to Ad-GFP)
D
40
10 （MOI）
30
**
10 0 Ad-GFP 2.5
αSMA protein level (relative to Ad-GFP)
1.0
*
*
**
0.5
0
Ad-GFP 2.5
5
7.5
Ad-sEH
10
OPN protein level (relative to Ad-GFP)
G 1.5
**
*
20
GAPDH
F
Ad-sEH
Ad-GFP
Time (hours)
5
7.5
10
Ad-sEH 2.5 2.0 1.5
*
**
**
**
Fig. 5. sEH overexpression promotes VSMC migration. A and B: confluent human VSMC monolayers were infected with Ad-GFP or Ad-sEH at 7.5 multiplicity of infection (MOI) for 24 h before scratch wounding. A: 14,15-EET and 14,15-DHET were determined with liquid chromatography coupled with tandem mass spectrometry (LC/MSMS) and the ratio of DHET to EET was calculated. B: scratch assay was performed and images were acquired at 0, 6, 12, 18, and 24 h. Data were analyzed by ANOVA, n ⫽ 5, **P ⬍ 0.01 compared with control. C: human VSMCs infected with adenovirus-mediated green fluorescent protein (Ad-GFP) or adenovirus-sEH (Ad-sEH) at 7.5 multiplicity of infection (MOI) for 24 h were seeded in the upper chamber of transwell plates. Magnification is ⫻400. The number of migrated cells was quantified in 5 random fields. Data are means ⫾ SD analyzed by t-test, n ⫽ 5, **P ⬍ 0.01. D–G: Western blot analysis of protein level of sEH, ␣-SMA, osteopontin (OPN), and GAPDH in rat VSMCs. Data are representative of 5 experiments.*P ⬍ 0.05, **P ⬍ 0.01 vs. Ad-GFP.
1.0 0.5 0
Ad-GFP 2.5
5
7.5
10
Ad-sEH
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
H1900
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
sEH promoted the migration of VSMCs. We next investigated the function of sEH in the migration of VSMCs. In vitro scratch wound assay revealed that PDGF-BB-stimulated human VSMC migration could be reversed by addition of the sEH inhibitor TUPS and 14,15-EET (Fig. 4A). When compared with PDGF-BB treatment alone, TUPS treatment reduced the mean migration distance at 6 and 12 h after scratch wounding to 72 and 76% in human VSMCs, respectively (Fig. 4B). In addition, with transwell assay and PDGF-BB used as a chemoattractant, PDGF-BB-induced VSMC migration was reduced to 66 and 60% by TUPS and 14,15-EET, respectively, at 6 h in human VSMCs (Fig. 4, C and D). Similar results were obtained in rat primary cultured VSMCs (data not shown). The effect of sEH may be dependent on levels of EETs; thus we measured the ratio of 14,15-DHET/EET with ultra-high-performance LC-MS/MS after treatment with PDGF in human VSMCs. Indeed, treatment with 20 ng/ml of PDGF for 24 h increased the ratio of EET to DHET, whereas TUPS reversed the effect of PDGF on EET degradation (Fig. 4E).
To further characterize sEH function in VSMCs, human and rat primary VSMCs were infected with Ad-GFP or Ad-sEH, and migration distance was investigated. In Ad-sEH-infected human VSMCs, the ratio of EET to DHET increased about onefold (Fig. 5A), and the mean migration distance was increased to 1.80-, 1.54-, and 1.50-fold compared with Ad-GFPinfected cells at 6, 12, and 24 h, respectively, after scratch wounding (Fig. 5B). Moreover, transwell assay revealed increased migration activity with Ad-sEH transfection at 6 h in humans (Fig. 5C). A similar trend was also observed in rat VSMCs (data not shown). As the fundamental mechanism of VSMC migration, VSMCs undergo phenotypic switching in response to injury, along with loss of expression of VSMC differentiation markers (12). To gain insight into the effect of sEH on VSMC phenotype, we examined the expression of differentiation markers in VSMCs transfected with Ad-sEH. Overexpression of sEH switched VSMCs from a contractile to synthetic phenotype, as evidenced by an increased level of OPN and decreased level of ␣-SMA (Fig. 5, D–G).
A
Day 3
Sham Ctrl
TUPS
Ctrl
TUPS
a
b
c
d
e
f
g
h
i
j
25
30
Media area (104 μm2)
20 15
**
10 5
0
D Neointima/Media ratio
C
Ctrl TUPS
1.0
Day 3 Ctrl TUPS
10
E
0.6
**
0.4
0.0
20
** Day 3
0
Day 7
0.8
0.2
Ctrl TUPS
*
Day 7
Circumference of EEL (mm)
B Neointimal area (104 μm2)
Fig. 6. Neointima formation in injured carotid arteries attenuated by TUPS. Rats underwent carotid-artery balloon injury. A: representative H&E staining of carotid arteries with sham surgery (left) or day 3 (middle) and day 7 (right) after balloon injury (⫻100) (a–e) and higher amplification (⫻400) (f–j). B–E: quantification of neointima area, media area, ratio of neointima to media, and circumference of external elastic lamina (EEL) in H&E-stained sections. Data are means ⫾ SD, n ⫽ 7–9 per group, *P ⬍ 0.05, **P ⬍ 0.01.
Day 7
5
Day 3
Day 7
Ctrl TUPS
4 3 2 1
0
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
Day 3
Day 7
H1901
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
sEH inhibition attenuated neointima formation in injured carotid arteries. Migration of VSMCs plays a key role in the development of neointima thickening. We next examined whether sEH inhibition could reduce intimal hyperplasia in vessels after carotid-artery balloon injury in rats. Male rats received TUPS (0.65 mg·kg⫺1·day⫺1) or vehicle by drinking water for 7 days, followed by carotid-artery injury. As an sEH inhibitor, TUPS was able to dramatically reduce sEH activity in rats (4). H&E staining demonstrated that the neointima area of arteries was reduced to 65 and 44% by TUPS treatment on days 3 and 7, respectively (Fig. 6, A and B), whereas the media area was intact (Fig. 6C). Accordingly, the ratio of neointima to media area was lower with TUPS than vehicle treatment on both days 3 and 7 (Fig. 6D). However, the circumference of the external elastic lamina was comparable between the two treatments (Fig. 6E). Thus inactivation of sEH was sufficient to attenuate neointima formation after balloon injury. Immunofluorescence double staining for sEH and ␣-SMA showed significantly increased sEH protein expression in VSMCs in the media and neointima of injured arteries on day 3 compared with that of the sham-operated control group, which was decreased by TUPS (Fig. 7, A and B). However, the sEH level was not further increased on day 7, which implied that the upregulation of sEH happened at an early stage (Fig. 7B). In
A
a-SMA
sEH
Nuclear
addition, the VSMCs had switched from a synthetic to a contractile phenotype by TUPS on days 3 and 7, as evidenced by decreased level of OPN (Fig. 7, C and D). DISCUSSION AND CONCLUSIONS
sEH enzymatically hydrolyzes the conversion of EETs and other fatty acid epoxides to the less active corresponding diols (22). EETs, derived from arachidonic acid by epoxygenation through cytochrome-P450 mono-oxygenases, have multiple biological functions, but difficulties arise with long-term EET treatment because they are rapidly metabolized and incorporated into membranes. Therefore, considerable interest has arisen in developing methods to enhance the bioavailability of EETs by inhibiting sEH. As expected, inhibition of sEH prevents several cardiovascular diseases, including high blood pressure, cardiac fibrosis, inflammation, and atherosclerosis (16, 19, 24). Therefore, the enzyme activity of sEH appears to play an important role in the development of cardiovascular disease. However, evidence of sEH expression and its role in humans is lacking. In our study, we found that 1) sEH protein was highly expressed in VSMCs of human coronary atherosclerotic arteries; 2) PDGF-BB posttranscriptionally upregulated sEH expression in rat primary VSMCs by stabilizing sEH Merge
C
Merge
DAPI
OPN
Day 3
Sham
Ctrl
TUPS
Day 7
Ctrl
2.0
Ctrl TUPS
1.5 1.0 0.5 0
Day 3
Day 7
20
D
OPN protein (relative to Sham)
B
Relative protein level of sEH
TUPS
Ctrl TUPS
15
* 10
**
5
0
Day 3
Day 7
Fig. 7. Expression of sEH was increased by carotid-artery balloon injury in rat vessel VSMCs. Rats were pretreated with vehicle or TUPS for 7 days and then underwent carotid-artery balloon injury. The results were analyzed on days 3 or 7. A: immunofluorescence double staining for sEH (green) and ␣-SMA (red) in VSMCs in media and neointima of injured arteries. B: quantification of the sEH density, which was normalized to that of sham-operated control. OPN expression was measured (C) and quantified (D); n ⫽ 7–9, *P ⬍ 0.05 and **P ⬍ 0.01, TUPS vs. control on day 3 or day 7. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
H1902
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
protein; 3) overexpression of sEH promoted the migration and phenotype switching in rat primary VSMCs, which could be reversed by the sEH inhibitor TUPS; and 4) sEH protein expression was increased in balloon-injured carotid arteries of rats, and TUPS attenuated the neointima formation. Although sEH is expressed in many organs (8), sEH protein levels in human coronary atherosclerotic lesions have not been reported. We found more sEH-positive sections in plaques of patients with than without coronary atherosclerosis. Moreover, the sEH-positive cells were located in VSMC-marker-positive areas, which implied that the high expression of sEH may play a role in the development of atherosclerosis via VSMCs. Migration of VSMCs, which occurs under the influence of basic fibroblast growth factor and inflammatory factors, plays an essential role during the progression of cardiovascular diseases such as atherosclerosis and restenosis (11). sEH was previously reported to be involved in regulating VSMC proliferation (7, 14). We extended these findings and found that the sEH inhibitor TUPS reversed the migration of primary rat VSMCs induced by PDGF-BB. Moreover, the overexpression of sEH promoted the migration and phenotype switching of primary VSMCs. EETs were shown to possess antimigratory effects on VSMCs through the cAMP-protein kinase A pathway, which might explain the effect of sEH on VSMC migration (17). In our study, we found that PDGF-BB upregulated sEH and promoted EET degradation, which supported the role for EETs in VSMC migration. sEH has many regulatory mechanisms at the transcription and protein levels. sEH expression can be regulated by 15dPGJ2, homocysteine, angiotensin II, and tyrosine nitration, as well as by peroxisome proliferator-activated receptor-␥ agonists (2, 5, 6, 18, 24). Our previous studies showed that sEH upregulation contributes to hypertension and cardiac hypertrophy (3, 23). In the present study, PDGF-BB induced sEH expression in dose- and time-dependent manners. However, our quantitative real-time RT-PCR and promoter deletion transfection data in rat primary VSMCs showed that PDGF-BB regulated sEH posttranscriptionally via stabilizing sEH protein. Although inhibition of sEH prevents VSMC migration, the effects of sEH inhibition on vascular remodeling in vivo were controversial. In spontaneously hypertensive stroke-prone rats, sEH inhibition or Ehpx2 deficiency reduced the inward remodeling in a common carotid ligation model rather than wireinduced vascular injury in femoral arteries, which indicated the mechanisms are endothelium dependent (15). However, Ehpx2 deficiency was found to attenuate neointima formation in a femoral artery cuff model but not following carotid artery ligation in apoE⫺/⫺ mice (14). In addition, sEH inhibitor is able to attenuate the development of atherosclerosis and abdominal aortic aneurysm formation but not ligation-induced carotid artery remodeling in apoE⫺/⫺ mice with angiotensin II infusion (25). Those two studies suggested that the antivascular remodeling effect of sEH inhibitor and Ehpx2 deficiency in hyperlipidemia models is dependent on lowering lipid level. In our study, we performed the artery injury surgery in the carotid artery of rat with normal lipid level. The discrepancy between our study and that of Simpkins et al. (15) might be caused by species specificity. Elucidating the mechanism controlling VSMC migration is critical for understanding the pathogenesis and progression of cardiovascular diseases. Our finding provides evidence that
sEH is involved in the progression of neointima formation by promoting VSMC migration, which suggests that sEH inhibition may be a potential therapeutic target for cardiovascular diseases. ACKNOWLEDGMENTS Y. Zhu is an investigator in the Collaborative Innovation Center of Beijing for Cardiovascular Research. Present address of L. Huo: Department of Pathology, Guangdong Women and Children’s Hospital, Guangzhou Medical College, Guangzhou, China. Present address of H. Su: College of Forensic Medicine, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061, PR China. GRANTS This study was supported in part by Major National Basic Research Grant of China 2012CB517500 and by National Natural Science Foundation of China Grants 81121061, 81130002, and 81322006. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Y.Z. conception and design of research; Q.W., J.H., W.D., and H.S. performed experiments; L.H. analyzed data; D.T. and B.H. interpreted results of experiments; Q.W. prepared figures; D.A. drafted manuscript; C.W., B.H., and D.A. edited and revised manuscript; Y.Z. and D.A. approved final submission. REFERENCES 1. Ahlskog JK, Dumelin CE, Trussel S, Marlind J, Neri D. In vivo targeting of tumor-associated carbonic anhydrases using acetazolamide derivatives. Bioorg Med Chem Lett 19: 4851–4856, 2009. 2. Ai D, Fu Y, Guo D, Tanaka H, Wang N, Tang C, Hammock BD, Shyy JY, Zhu Y. Angiotensin II up-regulates soluble epoxide hydrolase in vascular endothelium in vitro and in vivo. Proc Natl Acad Sci USA 104: 9018 –9023, 2007. 3. Ai D, Pang W, Li N, Xu M, Jones PD, Yang J, Zhang Y, Chiamvimonvat N, Shyy JY, Hammock BD, Zhu Y. Soluble epoxide hydrolase plays an essential role in angiotensin II-induced cardiac hypertrophy. Proc Natl Acad Sci USA 106: 564 –569, 2009. 4. Althurwi HN, Tse MM, Abdelhamid G, Zordoky BN, Hammock BD, El-Kadi AO. Soluble epoxide hydrolase inhibitor, TUPS, protects against isoprenaline-induced cardiac hypertrophy. Br J Pharmacol 168: 1794 – 1807, 2013. 5. Barbosa-Sicard E, Fromel T, Keseru B, Brandes RP, Morisseau C, Hammock BD, Braun T, Kruger M, Fleming I. Inhibition of the soluble epoxide hydrolase by tyrosine nitration. J Biol Chem 284: 28156 –28163, 2009. 6. Charles RL, Burgoyne JR, Mayr M, Weldon SM, Hubner N, Dong H, Morisseau C, Hammock BD, Landar A, Eaton P. Redox regulation of soluble epoxide hydrolase by 15-deoxy-delta-prostaglandin J2 controls coronary hypoxic vasodilation. Circ Res 108: 324 –334, 2011. 7. Davis BB, Thompson DA, Howard LL, Morisseau C, Hammock BD, Weiss RH. Inhibitors of soluble epoxide hydrolase attenuate vascular smooth muscle cell proliferation. Proc Natl Acad Sci USA 99: 2222–2227, 2002. 8. Enayetallah AE, French RA, Thibodeau MS, Grant DF. Distribution of soluble epoxide hydrolase and of cytochrome P450 2C8, 2C9, and 2J2 in human tissues. J Histochem Cytochem 52: 447–454, 2004. 9. Jovinge S, Hultgardh-Nilsson A, Regnstrom J, Nilsson J. Tumor necrosis factor-alpha activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol 17: 490 –497, 1997. 10. Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2: 329 –333, 2007. 11. Newby AC, Zaltsman AB. Molecular mechanisms in intimal hyperplasia. J Pathol 190: 300 –309, 2000.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION 12. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84: 767–801, 2004. 13. Regan CP, Adam PJ, Madsen CS, Owens GK. Molecular mechanisms of decreased smooth muscle differentiation marker expression after vascular injury. J Clin Invest 106: 1139 –1147, 2000. 14. Revermann M, Schloss M, Barbosa-Sicard E, Mieth A, Liebner S, Morisseau C, Geisslinger G, Schermuly RT, Fleming I, Hammock BD, Brandes RP. Soluble epoxide hydrolase deficiency attenuates neointima formation in the femoral cuff model of hyperlipidemic mice. Arterioscler Thromb Vasc Biol 30: 909 –914, 2010. 15. Simpkins AN, Rudic RD, Roy S, Tsai HJ, Hammock BD, Imig JD. Soluble epoxide hydrolase inhibition modulates vascular remodeling. Am J Physiol Heart Circ Physiol 298: H795–H806, 2010. 16. Spector AA, Fang X, Snyder GD, Weintraub NL. Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Prog Lipid Res 43: 55–90, 2004. 17. Sun J, Sui X, Bradbury JA, Zeldin DC, Conte MS, Liao JK. Inhibition of vascular smooth muscle cell migration by cytochrome p450 epoxygenase-derived eicosanoids. Circ Res 90: 1020 –1027, 2002. 18. Tanaka H, Kamita SG, Wolf NM, Harris TR, Wu Z, Morisseau C, Hammock BD. Transcriptional regulation of the human soluble epoxide hydrolase gene EPHX2. Biochim Biophys Acta 1779: 17–27, 2008. 19. Ulu A, Davis BB, Tsai HJ, Kim IH, Morisseau C, Inceoglu B, Fiehn O, Hammock BD, Weiss RH. Soluble epoxide hydrolase inhibitors reduce the development of atherosclerosis in apolipoprotein e-knockout mouse model. J Cardiovasc Pharmacol 52: 314 –323, 2008.
H1903
20. Wang L, Zheng J, Bai X, Liu B, Liu CJ, Xu Q, Zhu Y, Wang N, Kong W, Wang X. ADAMTS-7 mediates vascular smooth muscle cell migration and neointima formation in balloon-injured rat arteries. Circ Res 104: 688 –698, 2009. 21. Xu D, Li N, He Y, Timofeyev V, Lu L, Tsai HJ, Kim IH, Tuteja D, Mateo RK, Singapuri A, Davis BB, Low R, Hammock BD, Chiamvimonvat N. Prevention and reversal of cardiac hypertrophy by soluble epoxide hydrolase inhibitors. Proc Natl Acad Sci USA 103: 18733–18738, 2006. 22. Yu Z, Xu F, Huse LM, Morisseau C, Draper AJ, Newman JW, Parker C, Graham L, Engler MM, Hammock BD, Zeldin DC, Kroetz DL. Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids. Circ Res 87: 992–998, 2000. 23. Zhang D, Ai D, Tanaka H, Hammock BD, Zhu Y. DNA methylation of the promoter of soluble epoxide hydrolase silences its expression by an SP-1-dependent mechanism. Biochim Biophys Acta 1799: 659 –667, 2010. 24. Zhang D, Xie X, Chen Y, Hammock BD, Kong W, Zhu Y. Homocysteine upregulates soluble epoxide hydrolase in vascular endothelium in vitro and in vivo. Circ Res 110: 808 –817, 2012. 25. Zhang LN, Vincelette J, Cheng Y, Mehra U, Chen D, Anandan SK, Gless R, Webb HK, Wang YX. Inhibition of soluble epoxide hydrolase attenuated atherosclerosis, abdominal aortic aneurysm formation, and dyslipidemia. Arterioscler Thromb Vasc Biol 29: 1265–1270, 2009. 26. Zhang X, Yang N, Ai D, Zhu Y. Systematic metabolomic analysis of eicosanoids after omega-3 polyunsaturated fatty acid supplementation by a highly specific liquid chromatography-tandem mass spectrometry-based method. J Proteome Res 14: 1843–1853, 2015.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
Soluble epoxide hydrolase is involved in the development of atherosclerosis and arterial neointima formation by regulating smooth muscle cell migration Qingjie Wang,1,2* Leijun Huo,1* Jinlong He,3 Wenshuang Ding,1 Hang Su,1 Dongping Tian,1 Carrie Welch,4 Bruce D. Hammock,5 Ding Ai,3 and Yi Zhu3 1
Department of Pathology, Shantou University Medical College, Shantou, Guangdong, China; 2Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai, China; 3Department of Physiology and Pathophysiology, Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Medical University, Tianjin, China; 4Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York; and 5 Department of Entomology and Comprehensive Cancer Center, University of California, Davis, California Submitted 22 April 2015; accepted in final form 18 August 2015
Wang Q, Huo L, He J, Ding W, Su H, Tian D, Welch C, Hammock BD, Ai D, Zhu Y. Soluble epoxide hydrolase is involved in the development of atherosclerosis and arterial neointima formation by regulating smooth muscle cell migration. Am J Physiol Heart Circ Physiol 309: H1894 –H1903, 2015. First published October 9, 2015; doi:10.1152/ajpheart.00289.2015.—Epoxyeicosatrienoic acids (EETs) have beneficial effects on cardiovascular disease. Soluble epoxide hydrolase (sEH) metabolizes EETs to less active diols, thus diminishing their biological activity. sEH inhibitors can suppress the progression of atherosclerotic lesions in animal models. However, the regulation of sEH in vascular smooth muscle cells (VSMCs) and role of sEH in patients with atherosclerosis have not been evaluated. We hypothesize that sEH in VSMCs plays a pivotal role in atherosclerosis and injury-induced neointima formation. In this study, sEH expression in human autopsy atherosclerotic plaque was determined by immunohistochemistry. In cultured rat and human VSMCs, the phenotypic switching marker and sEH expression induced by plateletderived growth factor-BB (PDGF-BB) were examined by Western blot analysis. Carotid-artery balloon injury was performed after adenovirus-mediated overexpression of sEH or oral administration of a potent sEH inhibitor in Sprague-Dawley rats. sEH was highly expressed in VSMCs of the intima and media within human atherosclerotic plaque. In vitro, PDGF-BB upregulated the expression in VSMCs after transcription and promoted cell proliferation and migration; the latter effect could be largely attenuated by an sEH inhibitor. Adenovirus-mediated overexpression of sEH could mimic the effect of PDGF-BB and induce VSMC proliferation and migration. In vivo, the sEH inhibitor led to a significant decrease in injury-induced neointima formation in a rat carotid-artery injury model. These data establish the effect of sEH expression on atherosclerotic progression and vascular remodeling after injury, thus identifying a novel integrative role for sEH in VSMC phenotypic modulation and migration. Blocking sEH activity may be a potential therapeutic approach for ameliorating vascular occlusive disease. soluble epoxide hydrolase inhibitor; atherosclerosis; vascular smooth muscle cell; migration; neointima formation
NEW & NOTEWORTHY
In this study, we found that soluble epoxide hydrolase (sEH) was highly expressed in vascular smooth muscle cells (VSMCs) of human atherosclerotic plaque and in rat neointima. Inhibition of sEH largely attenuated platelet-derived growth factor* Q. Wang and L. Huo contributed equally to this work. Address for reprint requests and other correspondence: Y. Zhu, Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Medical Univ., Tianjin, 300070, China (e-mail: [email protected]). H1894
BB-induced VSMC migration in vitro and injury-induced neointima formation in vivo, which suggests that sEH inhibition may be a potential therapeutic target for cardiovascular diseases. EPOXYEICOSATRIENOIC ACIDS (EETs), derived from arachidonic acid, have several beneficial effects on cardiovascular homeostasis, including the ability to hyperpolarize vascular smooth muscle cells (VSMCs), dilate coronary arteries, and suppress adhesion molecules. Soluble epoxide hydrolase (sEH) metabolizes EETs to their corresponding dihydroxyeicosatrienoic acids, which limits the availability of EETs (16). Several models of atherosclerosis have suggested that inhibition of sEH, with an ensuing decrease in EET degradation, may attenuate atherosclerosis development and aneurysm formation (3, 14, 15, 19, 25). Treatment with sEH inhibitors also attenuated cardiohypertrophy in rats with angiotensin II treatment and in a murine model with transverse aortic constriction (21). However, the involvement of sEH in the development of human cardiovascular disease is unclear. VSMCs, a major blood vessel cell type, play major roles in maintaining vessel integrity and function. The migration of VSMCs from media to intima is pivotal for intimal thickening in cardiovascular disease. VSMCs in adult animals are plastic and retain their ability to switch rapidly from a contractile excitable phenotype to a migratory synthetic phenotype in response to injury (11). This modulation is the common fundamental biological mechanism of VSMC-related cardiovascular diseases such as atherosclerosis and neointimal formation after arterial injury. A characteristic of synthetic VSMCs is that they have low levels of differentiation marker proteins, including ␣-smooth muscle actin (␣-SMA) and smooth muscle protein 22␣ (SM22␣), which are required for the VSMC normal contractile function (12, 13). Given the important role of VSMCs in atherosclerosis and neointima formation after arterial injury and the effect of sEH/EETs on the cardiovascular system, we hypothesize that sEH in VSMCs plays a pivotal role in vascular remodeling. In the current study, we investigated the role of VSMC sEH in atherosclerotic and injury-induced neointima formation. We found that sEH was highly expressed in VSMCs of human atherosclerotic plaques. In vitro, sEH played a critical role in VSMC migration by inhibiting platelet-derived growth factor-BB (PDGF-BB)-induced differentiation. Pharmacological inhibition of sEH in a balloon-injured carotid artery model decreased neointima formation and injury-induced VSMCspecific gene expression. sH may be a potential therapeutic
0363-6135/15 Copyright © 2015 the American Physiological Society
http://www.ajpheart.org
H1895
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
target for attenuating VSMC dedifferentiation and migration under pathological conditions.
Table 1. Age and gender information of autopsy samples Age
MATERIALS AND METHODS
Reagents. Cycloheximide (CHX) and Hoechst stain reagent were from Sigma-Aldrich (St. Louis, MO). PDGF was from R&D Systems (Minneapolis, MN). Primary antibodies for sEH (sc-25797), osteopontin (OPN, sc-21742), GAPDH, CD68 (sc-9139), and polyperoxidase-anti-mouse/rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against smooth muscle ␣-actin (AA132) were from Beyotime Institute of Biotechnology (Beijing, China), and DyLight 488-labeled goat anti-rabbit IgG (H⫹L) and DyLight 594labeled goat anti-mouse IgG (H⫹L) were from Zhongshan Golden Bridge (Beijing, China). Human samples and analysis. Autopsy case records from 2001 to 2010 at the Verification Centre of Forensic Medicine affiliated with Shantou University were reviewed, from which 97 formalin-fixed
Sex
Group
Range
Mean
Male
Female
Nondiseased vessel, n ⫽ 36 Atherosclerotic plaques, n ⫽ 61
18–60 24–77
32.93 ⫾ 10.50 46.18 ⫾ 13.47
20 58
16 3
paraffin embedded coronary or coronary artery sections were chosen and stained with hematoxylin and eosin (H&E) to evaluate the degree of atherosclerosis. On the basis of H&E staining, sections were separated into two groups: one nondiseased and one with atherosclerotic plaque. Ponceau/Victoria blue (P/VB) was used to stain the elastic fibers surrounding vessels and proteins in cells, respectively. Sixty-one samples exhibited atherosclerotic plaques and 36 showed nonplaque arteries. The age and sex of individuals in each group are listed in Table 1. Analysis of sEH location and intensity was per-
H&E staining
A
Atherosclerotic plaques
Non-diseased vessels
B
f
C
P/VB
c
d
e
f
**
3
h
α-SMA
sEH b
4
CD68 d
g
a
Relative protein level of sEH
D
c
b
e
α-SMA
sEH
P/VB a
Fig. 1. Soluble epoxide hydrolase (EH) protein level is upregulated in atherosclerotic lesions of human coronary arteries. A: hematoxylin and eosin (H&E) staining of human coronary artery sections (nondiseased vessel, n ⫽ 36; atherosclerotic plaque, n ⫽ 61). B: representative immunohistochemical analysis of atherosclerotic patient coronary artery cross sections; a–d: immunostaining with Ponceau/ Victoria blue (P/VB) and antibodies for soluble EH (sEH), ␣-smooth muscle actin (␣-SMA), and CD68 (bar ⫽ 500 m); e–h: higher amplification of boxes (bar ⫽ 100 m). C: immunohistochemical staining with P/VB, sEH (arrows) and ␣-SMA. Top, bar ⫽ 100 m; bottom, higher magnification of boxes, bar ⫽ 50 m. D: quantification of sEH in intima and media. Nondiseased vessels, n ⫽ 36; atherosclerotic plaque, n ⫽ 61; data are means ⫾ SE, **P ⬍ 0.001, media of nondiseased vessel group vs. media of atherosclerotic plaque group. Bar ⫽ 100 m.
Non-diseased vessel group Atherosclerotic plaque group
2 1 0
Intima
Media
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
H1896
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
formed in a blinded fashion. The average density (integral optical density/area) was measured with Image-Pro Plus 6.0, and the result was determined as the sum of five different fields of each section. The level of sEH average density was normalized to that of nondiseased vessels in intima. The study was carried out in accordance with the Declaration of Helsinki and The Belmont Report (Washington, DC: U.S. Department of Health &Human Services, 1979), and was approved by the Ethics Committee of Shantou University Medical College. Animal artery injury. Male Sprague-Dawley rats (210 to 230 g) were housed in a temperature-controlled room with a 12-h:12-h light-dark cycle and had free access to standard chow and water. The sEH inhibitor 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) was designed and synthesized in Bruce Hammock’s laboratory at Columbia University in New York. When compared with early generations of competitive sEH inhibitors, TUPS exhibits high potency (IC50 ⫽ 7.0 nM in rat), excellent oral availabil-
A
ity, and pharmacokinetic properties (1). TUPS was dissolved and delivered at 0.65 mg·kg⫺1·day⫺1 in drinking water for 10 or 14 days as was previously reported (3), starting 7 days before surgery. A vehicle was administered to the control group. At 3 or 7 days after carotid artery injury, all animals (n ⫽ 7 to 9 per group) were anesthetized by CO2, and arteries were harvested. The protocols in the investigation followed those in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Publication No. 85-23, Revised). All animal experimental protocols were approved by the Shantou University Institutional Animal Care and Use Committee. Surgery was performed as described (20). Briefly, rats were anesthetized by injection of pentobarbital sodium (30 mg/kg ip). A balloon catheter of 1.5 mm in diameter (Medtronic, Minneapolis, MN) was introduced through the left external carotid artery and advanced 4 cm toward the thoracic aorta. The balloon was distended and pulled back to the bifurcation with constant rotation. This procedure was repeated two more times to ensure complete endothelial denudation. Contralat-
B
PDGF (24hr) 0
5
10
20
** 2
*
**
1
0
D
4
Human VSMC **
3 2
**
**
**
1 0 0
5
10
20
40 (ng/ml)
12h
24h
2.5 2.0
0.5
** **
**
**
** **
**
*
0.5 0
Human VSMC
2.5
*
2.0
*
1.5
**
**
1.0 0.5
** **
**
1.0
0 0
F
**
*
1.5
4
8
12
24 (h)
6
sEH-LUC activty
Relative mRNA level
Relative protein level of sEH Relative protein level of sEH
sEH relative protein level Relative protein level of sEH
3
1.0
0
8h
GAPDH
E 1.5
PDGF (ng/ml)
4h
sEH
GAPDH
C
0h
40 (ng/ml)
sEH
Fig. 2. Platelet-derived growth factor (PDGF) induces sEH expression in rat and human vascular smooth muscle cells (VSMCs). Rat VSMCs were treated with PDGF-BB for 24 h at the indicated dose (A) or 20 ng/ml PDGF for different times (B). Western blot analysis of sEH protein expression and quantification. *P ⬍ 0.05, **P ⬍ 0.01 compared with control. A paired t-test was performed. Human VSMCs were treated with PDGF-BB for 24 h at the indicated dose (C) or 20 ng/ml of PDGF for different times (D). Quantification of sEH protein expression is shown. *P ⬍ 0.05, **P ⬍ 0.01 compared with control. A paired t-test was performed. E: rat VSMCs were incubated with PDGF-BB at the indicated dose for 24 h. RT-PCR analysis of mRNA levels of sEH and VSMC differentiation markers was performed. Messenger RNA levels were normalized to those of GAPDH. *P ⬍ 0.05, **P ⬍ 0.01 compared with control. A t-test was performed. F: rat VSMCs were transfected with human sEH promoter-Luc plasmids (⫺1,000 and ⫺471 bp) and treated with 20 ng/ml of PDGF-BB for 24 h. -Gal plasmid was used as an internal control. Data are means ⫾ SD of 3 independent experiments.
PDGF (20ng/ml)
Ctrl PDGF 4
2
**** 0
0 5 10 20 40
0 5 10 20 40
sEH
-SMA
0 5 10 20 40 0 5 10 20 40
SM-22α
Calponin 1
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
sEH-1000
sEH-471
H1897
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
In vitro scratch assay and transwell migration assay. For scratchinduced wound healing assay, rat or human VSMCs were cultured until they were 90% confluent. The monolayers were scratched with use of a p200 pipette tip as described (10) and pretreated with TUPS (1 M) or 14,15-EET (100 nM) for 30 min and then incubated with 20 ng/ml of PDGF-BB or PBS for 24 h in serum-free medium. The farthest distance that cells migrated from the wound edge was measured (average of 5 independent microscope fields for each of 3 independent experiments). Analysis of VSMC invasion involved a modified Boyden chamber coated with an 8-m barrier of reconstituted basement-membrane protein similar in composition to that surrounding VSMCs in vivo (Millipore, Billerica, MA). Assays of sEH activity. EET and 14,15-dihydroxyeicosatrienoic acid (DHET) were measured as previously reported (26). Briefly, cell lysate samples were spiked with 1 l of internal standard mixture (5 ng for each internal standard). The Waters Oasis-HLB cartridges (1 ml volume) were used for solid-phase extraction. The elutions were further dried and redissolved in 30% acetonitrile. 14,15-EET and its metabolite of sEH, 14,15-DHET, were measured with ultra-highperformance liquid chromatography (UPLC; Waters, Milford, MA) and a 5500 QTRAP mass spectrometer (AB Sciex, Foster City, CA) equipped with a Turbo Ion Spray electrospray ionization source (liquid chromatography coupled with tandem mass spectrometry; LC-MS/MS) as described previously (26). The DHET-to-EET ratio was used to indicate sEH activity. Statistical analysis. Results were analyzed by two-tailed Student’s t-test or one-way or a two-way ANOVA or linear regression as indicated, by use of GraphPad Prism software. A Bonferroni test was used to test for significant post hoc differences revealed by ANOVA. A Pearson 2 test was used to compare sEH positivity in the intima and media of nondiseased and atherosclerotic tissue by use of SPSS v11.5 (SPSS Inc, Chicago, IL). P ⬍ 0.05 was considered statistically significant.
A
Ctrl CHX (h)
0
1
2
PDGF
4
8
4
5
0
1
2
4
8
sEH GAPDH
B
0.5
0.0 1
Ln[(sEH) t/(sEH) 0]
eral carotid arteries underwent a similar operation without injury and served as sham-operated controls. Morphometric analysis of rat carotid arteries. Rat arteries were perfusion-fixed with 4% paraformaldehyde. Briefly, cryosections (6 m thick, 350 m apart) were taken from the middle portion of the balloon-injured segment, and eight sections of each sample were analyzed by H&E staining and with Spot Image software (Diagnostic Instruments, Australia). The intima and media areas and the circumference of the external elastic lamina were determined, and the ratio of intima to media area was calculated. VSMC culture. Rat VSMCs were isolated from thoracic aortas of male Sprague-Dawley rats (180 to 200 g) as described (9) and maintained in DMEM (Gibco) containing 20% fetal bovine serum (FBS) (Highclone). Rats were euthanized with an overdose of pentobarbital sodium (100 mg/kg ip) before the aorta was excised. Purity of VSMCs was verified by anti-␣-actin antibody staining, and cells between passages 2 to 4 were made quiescent by incubating in serum-free DMEM for 48 h at 37°C in a humidified atmosphere containing 5% CO2 for in vitro experiments unless stated otherwise. A human VSMC cell line (HA-VSMC) was purchased from FuDan IBS Cell Center and cultured in DMEM/F12 with 10% FBS (Gibco), 0.05 mg/ml vitamin C (Sigma), 0.03 g/ml EGF (PrimeGene Bio Tech, Shanghai, China), 0.01 mg/ml insulin (Sigma), 0.01 mg/ml transferrin (Sigma), and 10 ng/ml sodium selenite (Sigma). Adenovirus infection. The protocol of VSMCs infected with adenovirus sEH (Ad-sEH) or adenovirus-mediated green fluorescent protein (Ad-GFP) has been described elsewhere (3, 18, 23). The adenovirus was added to the medium and incubated for 24 h. The infection efficiency was detected by RT-PCR and Western blot analysis. Ad-GFP was used as a negative control. Quantitative RT-PCR. Total RNA was extracted by use of TRIzol (Invitrogen, Carlsbad, CA). RNA concentration and quality were determined by spectral absorption at 260 and 280 nm. Equal amounts of total RNA were reverse transcribed by use of the AMV Reverse Transcription System (Promega, Madison, WI). Quantitative RT-PCR of cDNA involved the Brilliant SYBR Green QPCR system, with GAPDH as an internal control. The rat nucleotide primer sequences were 5=-GCTCTCAAAAAGAAAGGATT-3= and 5=-TGACAGGACTCTATGAGGAA-3= for sEH, 5=-AGCATCCGACCTTG-3= and 5=-AGCATAGCCCTCATAG-3= for ␣-SMA, 5=-ACATGTTCCAGACTGTTGAC-3= and 5=-TGTCTGTGAACTCCCTCTTA-3= for SM22␣, 5=-GTCTGCTGAGGTAAAGAACA-3= and 5=-ACTCATTGACCTTCTTCACA-3= for Cnn1, and 5=-TCCCTCAAGATTGTCAGC-3= and 5=-AGCCCTTCCACGATGC-3 for GAPDH. Western blot analysis. VSMCs were washed with PBS and lysed in RIPA buffer supplemented with protease inhibitor cocktail (NovasyGen, China). Protein concentration was determined by the bicinchoninic acid protein assay kit (NovasyGen). Equal amounts of protein were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Germany), which were incubated with the primary antibodies for sEH, ␣-SMA, OPN, and GAPDH, then corresponding secondary antibodies; blots were developed by use of Super ECL Plus Detection Reagent (Applygen, China). Transient transfection and luciferase activity assay. Plasmids of the human sEH promoter, sEH-1000bp-Luc and sEH-471bp-Luc, were used for transient transfection. VSMCs at 80% confluence were transfected with 1 g of sEH promoter and 0.5 g of CMV-galactosidase by use of a Jet PEI (Polyplus, San Marcos, CA) for 24 h. Cells were treated with 20 ng/ml of PDGF-BB for another 24 h, and then lysed and collected for luciferase activity assay (Luciferase Reporter Assay System, Promega). Protein half-life measurements. VSMCs were pretreated with CHX (100 g/ml) for 30 min and then incubated with or without PDGF (20 ng/ml). Cells were harvested at 0, 1, 2, 4, and 8 h, and the amount of protein was detected by Western blot analysis. The kinetic constant and half-life of sEH protein with and without PDGF-BB were determined by linear regression analysis.
2
3
6
7
8
Time (h)
Time (h) -0.5
PDGF+CHX -1.0
K= -0.15± 0.01 t1/2 =4.60± 0.15 -1.5
-2.0
CHX K= -0.24± ± 0.01 t1/2 =2.47± 0.50
-2.5
Fig. 3. PDGF increases the stabilization of sEH protein in rat VSMCs. A: VSMCs were serum-starved for 48 h, then treated with cycloheximide (CHX) (100 g/ml) for the indicated time, with or without PDGF-BB (20 ng/ml). Western blot analysis of sEH protein level was normalized to GAPDH. Data are representative of 5 experiments. B: the slope of the decay line was calculated by standard linear regression analysis (P ⬍ 0.0001).
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
H1898
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
A
B
12h
Ctrl
4
PDGF
PDGF
Ctrl
**
PDGF+TUPS PDGF+EET
PDGF+EET
Relatived Migration Distance
3
PDGF+TUPS
** # * ##
** 2
**
##
** # 1
0 0
5
D
C Ctrl
10
15
Time (hours)
PDGF 40
PDGF+EET
Cell Number
30
PDGF+TUPS
**
20
** #
* ##
10 0
E
2.0
DHET/EET ratio (fold of control)
1.5
* #
1.0
0.5
0.0
Fig. 4. Migration of human VSMCs induced by PDGF was partly reversed by the sEH inhibitor 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoro-methoxyphenyl)-urea (TUPS) or epoxyeicosatrienoic acid (14,15-EET). A and B: confluent human VSMC monolayers were preincubated with TUPS (1 M) or 14,15-EET (100 nM) for 30 min and then treated with 20 ng/ml of PDGF. Scratch assay was performed. Images were taken at ⫻100 magnification. Data are means ⫾ SD of 5 experiments. *P ⬍ 0.05, **P ⬍ 0.01 compared with control; #P ⬍ 0.05 compared with PDGF alone. C: human VSMCs were seeded in the upper chamber of transwells. PDGF-BB (20 ng/ml) was added in the lower chamber as a chemoattractant with TUPS (1 M) or 14,15-EET (100 nM). Magnification is ⫻400. D: the mean cell number migrating across the filter was quantified in five randomly chosen fields. E: human VSMCs were incubated with or without TUPS (1 M) and treated with 20 ng/ml PDGF for 24 h, after which 14,15-EET and 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) were determined with LC/MS-MS and the DHET-to-EET ratio was calculated. Data are means ⫾ SD and analyzed by ANOVA from 5 independent experiments. *P ⬍ 0.05, **P ⬍ 0.01 compared with control; #P ⬍ 0.05, ##P ⬍ 0.05 compared with PDGF alone. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
H1899
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION RESULTS
of sEH in response to the VSMC migration inducer PDGF-BB. Rat and human primary VSMCs were treated with different doses of PDGF-BB for 24 h. sEH protein expression was induced by PDGF-BB at 10 ng/ml and peaked at 40 ng/ml (Fig. 2, A and C). In addition, PDGF treatment increased sEH protein levels at 8 and 4 h, in rat and human cells, respectively, which lasted for at least 24 h (Fig. 2, B and D). Thus PDGF induced sEH expression in both a dose- and time-dependent manner. The increase in sEH expression paralleled that of a reduced expression of contractile SMC markers such as ␣-SMA, calponin 1, and SM22␣. However, the mRNA levels of sEH and luciferase activity of the sEH promoter were not changed by PDGF-BB in VSMCs (Fig. 2, E and F). Therefore, PDGF-BB-induced regulation of sEH occurred posttranscriptionally. Because sEH was regulated by PDGF at the protein level, we next tested whether PDGF-BB enhanced the stability of sEH protein. We used CHX to block protein synthesis and measured the degradation of sEH in VSMCs. Soluble degradation was slower with PDGF-BB than control treatment (Fig. 3A). CHX treatment produced an sEH half-life of 2.5 ⫾ 0.5 min in control cells and 4.6 ⫾ 0.2 min in PDGF-treated VSMCs (Fig. 3B).
sEH protein is highly expressed in VSMCs in human atherosclerotic lesions. To investigate the role of sEH in atherosclerosis, we studied its expression in human coronary artery sections with polyclonal rabbit anti-human sEH antibody. Sections from 97 autopsy cases were divided into two groups: sections containing nondiseased vessels and sections containing atherosclerotic plaques (Fig. 1A) according to H&E staining of samples and pathology diagnosis. To further identify the sEH-positive cells, we immunostained human sections with P/VB and antibodies for sEH, ␣-SMA, and CD68. The expression of sEH corresponded to that of ␣-SMA rather than CD68 (Fig. 1B), which suggests that sEH was predominantly expressed in VSMCs in the intima and media (Fig. 1C). Moreover, the density of sEH was comparable in intima, which was significantly increased in the media of atherosclerotic plaques (Fig. 1D), so the high sEH expression in the VSMCs of atherosclerotic intima might be related to the development of atherosclerosis. PDGF-BB posttranscriptionally upregulated sEH expression in rat primary and human VSMCs. To explore a potential role for sEH in VSMC migration, we measured the expression
B
**
DHET/EET ratio (fold of control)
2.0 1.5 1.0 0.5 0.0
Ad-GFP
C
Ad-GFP
5
30
Ad-sEH
*
3
**
2 1 0
Ad-sEH
**
*
4
Cell Number
2.5
Relatived Migration Distance
A
0
10
20
10
0
20
Ad-sEH
Ad-GFP 7.5
2.5
5
7.5
sEH α-SMA OPN
E sEH protein level (relative to Ad-GFP)
D
40
10 （MOI）
30
**
10 0 Ad-GFP 2.5
αSMA protein level (relative to Ad-GFP)
1.0
*
*
**
0.5
0
Ad-GFP 2.5
5
7.5
Ad-sEH
10
OPN protein level (relative to Ad-GFP)
G 1.5
**
*
20
GAPDH
F
Ad-sEH
Ad-GFP
Time (hours)
5
7.5
10
Ad-sEH 2.5 2.0 1.5
*
**
**
**
Fig. 5. sEH overexpression promotes VSMC migration. A and B: confluent human VSMC monolayers were infected with Ad-GFP or Ad-sEH at 7.5 multiplicity of infection (MOI) for 24 h before scratch wounding. A: 14,15-EET and 14,15-DHET were determined with liquid chromatography coupled with tandem mass spectrometry (LC/MSMS) and the ratio of DHET to EET was calculated. B: scratch assay was performed and images were acquired at 0, 6, 12, 18, and 24 h. Data were analyzed by ANOVA, n ⫽ 5, **P ⬍ 0.01 compared with control. C: human VSMCs infected with adenovirus-mediated green fluorescent protein (Ad-GFP) or adenovirus-sEH (Ad-sEH) at 7.5 multiplicity of infection (MOI) for 24 h were seeded in the upper chamber of transwell plates. Magnification is ⫻400. The number of migrated cells was quantified in 5 random fields. Data are means ⫾ SD analyzed by t-test, n ⫽ 5, **P ⬍ 0.01. D–G: Western blot analysis of protein level of sEH, ␣-SMA, osteopontin (OPN), and GAPDH in rat VSMCs. Data are representative of 5 experiments.*P ⬍ 0.05, **P ⬍ 0.01 vs. Ad-GFP.
1.0 0.5 0
Ad-GFP 2.5
5
7.5
10
Ad-sEH
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
H1900
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
sEH promoted the migration of VSMCs. We next investigated the function of sEH in the migration of VSMCs. In vitro scratch wound assay revealed that PDGF-BB-stimulated human VSMC migration could be reversed by addition of the sEH inhibitor TUPS and 14,15-EET (Fig. 4A). When compared with PDGF-BB treatment alone, TUPS treatment reduced the mean migration distance at 6 and 12 h after scratch wounding to 72 and 76% in human VSMCs, respectively (Fig. 4B). In addition, with transwell assay and PDGF-BB used as a chemoattractant, PDGF-BB-induced VSMC migration was reduced to 66 and 60% by TUPS and 14,15-EET, respectively, at 6 h in human VSMCs (Fig. 4, C and D). Similar results were obtained in rat primary cultured VSMCs (data not shown). The effect of sEH may be dependent on levels of EETs; thus we measured the ratio of 14,15-DHET/EET with ultra-high-performance LC-MS/MS after treatment with PDGF in human VSMCs. Indeed, treatment with 20 ng/ml of PDGF for 24 h increased the ratio of EET to DHET, whereas TUPS reversed the effect of PDGF on EET degradation (Fig. 4E).
To further characterize sEH function in VSMCs, human and rat primary VSMCs were infected with Ad-GFP or Ad-sEH, and migration distance was investigated. In Ad-sEH-infected human VSMCs, the ratio of EET to DHET increased about onefold (Fig. 5A), and the mean migration distance was increased to 1.80-, 1.54-, and 1.50-fold compared with Ad-GFPinfected cells at 6, 12, and 24 h, respectively, after scratch wounding (Fig. 5B). Moreover, transwell assay revealed increased migration activity with Ad-sEH transfection at 6 h in humans (Fig. 5C). A similar trend was also observed in rat VSMCs (data not shown). As the fundamental mechanism of VSMC migration, VSMCs undergo phenotypic switching in response to injury, along with loss of expression of VSMC differentiation markers (12). To gain insight into the effect of sEH on VSMC phenotype, we examined the expression of differentiation markers in VSMCs transfected with Ad-sEH. Overexpression of sEH switched VSMCs from a contractile to synthetic phenotype, as evidenced by an increased level of OPN and decreased level of ␣-SMA (Fig. 5, D–G).
A
Day 3
Sham Ctrl
TUPS
Ctrl
TUPS
a
b
c
d
e
f
g
h
i
j
25
30
Media area (104 μm2)
20 15
**
10 5
0
D Neointima/Media ratio
C
Ctrl TUPS
1.0
Day 3 Ctrl TUPS
10
E
0.6
**
0.4
0.0
20
** Day 3
0
Day 7
0.8
0.2
Ctrl TUPS
*
Day 7
Circumference of EEL (mm)
B Neointimal area (104 μm2)
Fig. 6. Neointima formation in injured carotid arteries attenuated by TUPS. Rats underwent carotid-artery balloon injury. A: representative H&E staining of carotid arteries with sham surgery (left) or day 3 (middle) and day 7 (right) after balloon injury (⫻100) (a–e) and higher amplification (⫻400) (f–j). B–E: quantification of neointima area, media area, ratio of neointima to media, and circumference of external elastic lamina (EEL) in H&E-stained sections. Data are means ⫾ SD, n ⫽ 7–9 per group, *P ⬍ 0.05, **P ⬍ 0.01.
Day 7
5
Day 3
Day 7
Ctrl TUPS
4 3 2 1
0
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
Day 3
Day 7
H1901
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
sEH inhibition attenuated neointima formation in injured carotid arteries. Migration of VSMCs plays a key role in the development of neointima thickening. We next examined whether sEH inhibition could reduce intimal hyperplasia in vessels after carotid-artery balloon injury in rats. Male rats received TUPS (0.65 mg·kg⫺1·day⫺1) or vehicle by drinking water for 7 days, followed by carotid-artery injury. As an sEH inhibitor, TUPS was able to dramatically reduce sEH activity in rats (4). H&E staining demonstrated that the neointima area of arteries was reduced to 65 and 44% by TUPS treatment on days 3 and 7, respectively (Fig. 6, A and B), whereas the media area was intact (Fig. 6C). Accordingly, the ratio of neointima to media area was lower with TUPS than vehicle treatment on both days 3 and 7 (Fig. 6D). However, the circumference of the external elastic lamina was comparable between the two treatments (Fig. 6E). Thus inactivation of sEH was sufficient to attenuate neointima formation after balloon injury. Immunofluorescence double staining for sEH and ␣-SMA showed significantly increased sEH protein expression in VSMCs in the media and neointima of injured arteries on day 3 compared with that of the sham-operated control group, which was decreased by TUPS (Fig. 7, A and B). However, the sEH level was not further increased on day 7, which implied that the upregulation of sEH happened at an early stage (Fig. 7B). In
A
a-SMA
sEH
Nuclear
addition, the VSMCs had switched from a synthetic to a contractile phenotype by TUPS on days 3 and 7, as evidenced by decreased level of OPN (Fig. 7, C and D). DISCUSSION AND CONCLUSIONS
sEH enzymatically hydrolyzes the conversion of EETs and other fatty acid epoxides to the less active corresponding diols (22). EETs, derived from arachidonic acid by epoxygenation through cytochrome-P450 mono-oxygenases, have multiple biological functions, but difficulties arise with long-term EET treatment because they are rapidly metabolized and incorporated into membranes. Therefore, considerable interest has arisen in developing methods to enhance the bioavailability of EETs by inhibiting sEH. As expected, inhibition of sEH prevents several cardiovascular diseases, including high blood pressure, cardiac fibrosis, inflammation, and atherosclerosis (16, 19, 24). Therefore, the enzyme activity of sEH appears to play an important role in the development of cardiovascular disease. However, evidence of sEH expression and its role in humans is lacking. In our study, we found that 1) sEH protein was highly expressed in VSMCs of human coronary atherosclerotic arteries; 2) PDGF-BB posttranscriptionally upregulated sEH expression in rat primary VSMCs by stabilizing sEH Merge
C
Merge
DAPI
OPN
Day 3
Sham
Ctrl
TUPS
Day 7
Ctrl
2.0
Ctrl TUPS
1.5 1.0 0.5 0
Day 3
Day 7
20
D
OPN protein (relative to Sham)
B
Relative protein level of sEH
TUPS
Ctrl TUPS
15
* 10
**
5
0
Day 3
Day 7
Fig. 7. Expression of sEH was increased by carotid-artery balloon injury in rat vessel VSMCs. Rats were pretreated with vehicle or TUPS for 7 days and then underwent carotid-artery balloon injury. The results were analyzed on days 3 or 7. A: immunofluorescence double staining for sEH (green) and ␣-SMA (red) in VSMCs in media and neointima of injured arteries. B: quantification of the sEH density, which was normalized to that of sham-operated control. OPN expression was measured (C) and quantified (D); n ⫽ 7–9, *P ⬍ 0.05 and **P ⬍ 0.01, TUPS vs. control on day 3 or day 7. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
H1902
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION
protein; 3) overexpression of sEH promoted the migration and phenotype switching in rat primary VSMCs, which could be reversed by the sEH inhibitor TUPS; and 4) sEH protein expression was increased in balloon-injured carotid arteries of rats, and TUPS attenuated the neointima formation. Although sEH is expressed in many organs (8), sEH protein levels in human coronary atherosclerotic lesions have not been reported. We found more sEH-positive sections in plaques of patients with than without coronary atherosclerosis. Moreover, the sEH-positive cells were located in VSMC-marker-positive areas, which implied that the high expression of sEH may play a role in the development of atherosclerosis via VSMCs. Migration of VSMCs, which occurs under the influence of basic fibroblast growth factor and inflammatory factors, plays an essential role during the progression of cardiovascular diseases such as atherosclerosis and restenosis (11). sEH was previously reported to be involved in regulating VSMC proliferation (7, 14). We extended these findings and found that the sEH inhibitor TUPS reversed the migration of primary rat VSMCs induced by PDGF-BB. Moreover, the overexpression of sEH promoted the migration and phenotype switching of primary VSMCs. EETs were shown to possess antimigratory effects on VSMCs through the cAMP-protein kinase A pathway, which might explain the effect of sEH on VSMC migration (17). In our study, we found that PDGF-BB upregulated sEH and promoted EET degradation, which supported the role for EETs in VSMC migration. sEH has many regulatory mechanisms at the transcription and protein levels. sEH expression can be regulated by 15dPGJ2, homocysteine, angiotensin II, and tyrosine nitration, as well as by peroxisome proliferator-activated receptor-␥ agonists (2, 5, 6, 18, 24). Our previous studies showed that sEH upregulation contributes to hypertension and cardiac hypertrophy (3, 23). In the present study, PDGF-BB induced sEH expression in dose- and time-dependent manners. However, our quantitative real-time RT-PCR and promoter deletion transfection data in rat primary VSMCs showed that PDGF-BB regulated sEH posttranscriptionally via stabilizing sEH protein. Although inhibition of sEH prevents VSMC migration, the effects of sEH inhibition on vascular remodeling in vivo were controversial. In spontaneously hypertensive stroke-prone rats, sEH inhibition or Ehpx2 deficiency reduced the inward remodeling in a common carotid ligation model rather than wireinduced vascular injury in femoral arteries, which indicated the mechanisms are endothelium dependent (15). However, Ehpx2 deficiency was found to attenuate neointima formation in a femoral artery cuff model but not following carotid artery ligation in apoE⫺/⫺ mice (14). In addition, sEH inhibitor is able to attenuate the development of atherosclerosis and abdominal aortic aneurysm formation but not ligation-induced carotid artery remodeling in apoE⫺/⫺ mice with angiotensin II infusion (25). Those two studies suggested that the antivascular remodeling effect of sEH inhibitor and Ehpx2 deficiency in hyperlipidemia models is dependent on lowering lipid level. In our study, we performed the artery injury surgery in the carotid artery of rat with normal lipid level. The discrepancy between our study and that of Simpkins et al. (15) might be caused by species specificity. Elucidating the mechanism controlling VSMC migration is critical for understanding the pathogenesis and progression of cardiovascular diseases. Our finding provides evidence that
sEH is involved in the progression of neointima formation by promoting VSMC migration, which suggests that sEH inhibition may be a potential therapeutic target for cardiovascular diseases. ACKNOWLEDGMENTS Y. Zhu is an investigator in the Collaborative Innovation Center of Beijing for Cardiovascular Research. Present address of L. Huo: Department of Pathology, Guangdong Women and Children’s Hospital, Guangzhou Medical College, Guangzhou, China. Present address of H. Su: College of Forensic Medicine, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061, PR China. GRANTS This study was supported in part by Major National Basic Research Grant of China 2012CB517500 and by National Natural Science Foundation of China Grants 81121061, 81130002, and 81322006. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Y.Z. conception and design of research; Q.W., J.H., W.D., and H.S. performed experiments; L.H. analyzed data; D.T. and B.H. interpreted results of experiments; Q.W. prepared figures; D.A. drafted manuscript; C.W., B.H., and D.A. edited and revised manuscript; Y.Z. and D.A. approved final submission. REFERENCES 1. Ahlskog JK, Dumelin CE, Trussel S, Marlind J, Neri D. In vivo targeting of tumor-associated carbonic anhydrases using acetazolamide derivatives. Bioorg Med Chem Lett 19: 4851–4856, 2009. 2. Ai D, Fu Y, Guo D, Tanaka H, Wang N, Tang C, Hammock BD, Shyy JY, Zhu Y. Angiotensin II up-regulates soluble epoxide hydrolase in vascular endothelium in vitro and in vivo. Proc Natl Acad Sci USA 104: 9018 –9023, 2007. 3. Ai D, Pang W, Li N, Xu M, Jones PD, Yang J, Zhang Y, Chiamvimonvat N, Shyy JY, Hammock BD, Zhu Y. Soluble epoxide hydrolase plays an essential role in angiotensin II-induced cardiac hypertrophy. Proc Natl Acad Sci USA 106: 564 –569, 2009. 4. Althurwi HN, Tse MM, Abdelhamid G, Zordoky BN, Hammock BD, El-Kadi AO. Soluble epoxide hydrolase inhibitor, TUPS, protects against isoprenaline-induced cardiac hypertrophy. Br J Pharmacol 168: 1794 – 1807, 2013. 5. Barbosa-Sicard E, Fromel T, Keseru B, Brandes RP, Morisseau C, Hammock BD, Braun T, Kruger M, Fleming I. Inhibition of the soluble epoxide hydrolase by tyrosine nitration. J Biol Chem 284: 28156 –28163, 2009. 6. Charles RL, Burgoyne JR, Mayr M, Weldon SM, Hubner N, Dong H, Morisseau C, Hammock BD, Landar A, Eaton P. Redox regulation of soluble epoxide hydrolase by 15-deoxy-delta-prostaglandin J2 controls coronary hypoxic vasodilation. Circ Res 108: 324 –334, 2011. 7. Davis BB, Thompson DA, Howard LL, Morisseau C, Hammock BD, Weiss RH. Inhibitors of soluble epoxide hydrolase attenuate vascular smooth muscle cell proliferation. Proc Natl Acad Sci USA 99: 2222–2227, 2002. 8. Enayetallah AE, French RA, Thibodeau MS, Grant DF. Distribution of soluble epoxide hydrolase and of cytochrome P450 2C8, 2C9, and 2J2 in human tissues. J Histochem Cytochem 52: 447–454, 2004. 9. Jovinge S, Hultgardh-Nilsson A, Regnstrom J, Nilsson J. Tumor necrosis factor-alpha activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol 17: 490 –497, 1997. 10. Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2: 329 –333, 2007. 11. Newby AC, Zaltsman AB. Molecular mechanisms in intimal hyperplasia. J Pathol 190: 300 –309, 2000.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
ROLE OF sEH IN SMOOTH MUSCLE CELL MIGRATION 12. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84: 767–801, 2004. 13. Regan CP, Adam PJ, Madsen CS, Owens GK. Molecular mechanisms of decreased smooth muscle differentiation marker expression after vascular injury. J Clin Invest 106: 1139 –1147, 2000. 14. Revermann M, Schloss M, Barbosa-Sicard E, Mieth A, Liebner S, Morisseau C, Geisslinger G, Schermuly RT, Fleming I, Hammock BD, Brandes RP. Soluble epoxide hydrolase deficiency attenuates neointima formation in the femoral cuff model of hyperlipidemic mice. Arterioscler Thromb Vasc Biol 30: 909 –914, 2010. 15. Simpkins AN, Rudic RD, Roy S, Tsai HJ, Hammock BD, Imig JD. Soluble epoxide hydrolase inhibition modulates vascular remodeling. Am J Physiol Heart Circ Physiol 298: H795–H806, 2010. 16. Spector AA, Fang X, Snyder GD, Weintraub NL. Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Prog Lipid Res 43: 55–90, 2004. 17. Sun J, Sui X, Bradbury JA, Zeldin DC, Conte MS, Liao JK. Inhibition of vascular smooth muscle cell migration by cytochrome p450 epoxygenase-derived eicosanoids. Circ Res 90: 1020 –1027, 2002. 18. Tanaka H, Kamita SG, Wolf NM, Harris TR, Wu Z, Morisseau C, Hammock BD. Transcriptional regulation of the human soluble epoxide hydrolase gene EPHX2. Biochim Biophys Acta 1779: 17–27, 2008. 19. Ulu A, Davis BB, Tsai HJ, Kim IH, Morisseau C, Inceoglu B, Fiehn O, Hammock BD, Weiss RH. Soluble epoxide hydrolase inhibitors reduce the development of atherosclerosis in apolipoprotein e-knockout mouse model. J Cardiovasc Pharmacol 52: 314 –323, 2008.
H1903
20. Wang L, Zheng J, Bai X, Liu B, Liu CJ, Xu Q, Zhu Y, Wang N, Kong W, Wang X. ADAMTS-7 mediates vascular smooth muscle cell migration and neointima formation in balloon-injured rat arteries. Circ Res 104: 688 –698, 2009. 21. Xu D, Li N, He Y, Timofeyev V, Lu L, Tsai HJ, Kim IH, Tuteja D, Mateo RK, Singapuri A, Davis BB, Low R, Hammock BD, Chiamvimonvat N. Prevention and reversal of cardiac hypertrophy by soluble epoxide hydrolase inhibitors. Proc Natl Acad Sci USA 103: 18733–18738, 2006. 22. Yu Z, Xu F, Huse LM, Morisseau C, Draper AJ, Newman JW, Parker C, Graham L, Engler MM, Hammock BD, Zeldin DC, Kroetz DL. Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids. Circ Res 87: 992–998, 2000. 23. Zhang D, Ai D, Tanaka H, Hammock BD, Zhu Y. DNA methylation of the promoter of soluble epoxide hydrolase silences its expression by an SP-1-dependent mechanism. Biochim Biophys Acta 1799: 659 –667, 2010. 24. Zhang D, Xie X, Chen Y, Hammock BD, Kong W, Zhu Y. Homocysteine upregulates soluble epoxide hydrolase in vascular endothelium in vitro and in vivo. Circ Res 110: 808 –817, 2012. 25. Zhang LN, Vincelette J, Cheng Y, Mehra U, Chen D, Anandan SK, Gless R, Webb HK, Wang YX. Inhibition of soluble epoxide hydrolase attenuated atherosclerosis, abdominal aortic aneurysm formation, and dyslipidemia. Arterioscler Thromb Vasc Biol 29: 1265–1270, 2009. 26. Zhang X, Yang N, Ai D, Zhu Y. Systematic metabolomic analysis of eicosanoids after omega-3 polyunsaturated fatty acid supplementation by a highly specific liquid chromatography-tandem mass spectrometry-based method. J Proteome Res 14: 1843–1853, 2015.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00289.2015 • www.ajpheart.org
