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Abdominal aortic aneurysm (AAA) is an asymptomatic, potentially lethal disease whose ruptures have a high mortality rate. An effective pharmacological approach to decrease expansion or prevent the rupture of AAAs in humans remains lacking. Previous studies have suggested that activator protein 1 (c-Jun/AP-1) and C/EBP homologous protein (Chop) are involved in the development of AAA. The purpose of the present study was to investigate whether c-Jun/AP-1 mediates Chop overexpression in AAA. c-Jun/AP-1 and Chop protein levels were determined in an angiotensin II (Ang II)-induced AAA model using apolipoprotein E-deficient mice. Additionally, mouse aortic smooth muscle cells (MOVAS cell line) were treated with Ang II. Apoptosis was evaluated via TUNEL assay, MOVAS cell migration ability was assessed by monolayer wound healing assay and the levels of c-Jun/AP-1 and Chop were determined by western blotting, immunofluorescence and immunocytochemical assays. Following c-Jun silencing using c-Jun-specific small interfering (si)RNA, Chop expression was evaluated. Furthermore, chromatin immunoprecipitation (ChIP) was used to investigate whether c-Jun/Ap-1 binds directly to the DNA damage-inducible transcript 3 protein (Ddit3) promoter. It was observed that c-Jun/AP-1 and Chop were synchronously overexpressed in Ang II-induced AAA and Ang II-treated cells, and that apoptosis and migration were induced by Ang II. In addition, Chop was suppressed when c-Jun was silenced by targeted siRNA. Notably, the ChIP assay demonstrated that the DNA fragments pulled down by primary antibodies against c-Jun/Ap-1 were able to be amplified by (Ddit3) promoter-specific primers. c-Jun/AP-1 may therefore mediate Chop expression in MOVAS cells via Ddit3. These results suggested that c-Jun/AP-1 may be a novel target for AAA therapy.
Abdominal aortic aneurysm (AAA) involves chronic transmural inflammation and structural deterioration of the tissue architecture, leading to a progressively enlarged abdominal aorta. AAAs usually remain asymptomatic until a rupture occurs, which is associated with high morbidity and mortality rates in the adult human population (
Activator protein 1 (AP-1) is a dimeric transcription factor that controls gene expression, cell proliferation, differentiation and apoptosis (
C/EBP homologous protein (Chop), also termed GADD153, is a transcription factor and a member of the basic leucine zipper domain family (
In the present study, the molecular mechanisms underlying AAA development were evaluated with a focus on the roles of c-Jun/AP-1 and Chop, and it was determined that c-Jun/AP-1 is overexpressed in an Ang II-induced AAA model and Ang II-treated mouse aortic smooth muscle cell line (MOVAS cells). Chop was synchronously overexpressed; the regulation of Chop expression and apoptosis by c-Jun/AP-1 in AAA was then investigated. c-Jun/AP-1 may have had an essential role as a transcriptional regulator of Ddit3, resulting in Chop overexpression and the acceleration of AAA development.
All animals were housed at the animal care facility of Tongji Medical College, (Wuhan, China) under specific pathogen-free conditions and were fed a normal diet. Mice were housed in temperature-controlled cages (20±1°C, 55±5% humidity) with a 12 h light-dark cycle and given free access to water and food. All animal experiments were performed in accordance with the Animal Research Reporting of
A total of 30 10-week-old apolipoprotein E-deficient (ApoE−/−; C57BL/6 background) male mice (~20 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). Mice were randomly divided into 2 groups (n=15 per group) and implanted with osmotic pumps (Model 2004; Durect Corporation, Cupertino, CA, USA) containing either Ang II (1,000 ng/kg/min; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or saline (sham) for 4 weeks (
Aortas were fixed in 4% paraformaldehyde dissolved in PBS at room temperature for 24 h and embedded in paraffin for histological analyses. Serial cross-sections (3 µm) were produced from the aorta. For the morphometric analysis, the sections were stained with hematoxylin and eosin at room temperature. Briefly, the sections were incubated with hematoxylin for 5–10 min following rehydration, washed with 1% ethanol hydrochloride for 5 sec, and stained with eosin for 3 min.
The aorta specimens were prepared as described above. The arterial sections were blocked in PBS with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at room temperature for 1 h, and incubated overnight at 4°C with primary antibodies against c-Jun/Ap-1 (cat. no. 711202; dilution, 1:200; Invitrogen; Thermo Fisher Scientific, Inc.) and α-smooth muscle actin (α-SMA; cat. no. BM0002; dilution, 1:50; Boster Biological Technology, Pleasanton, CA, USA). Antibodies were replaced with secondary antibodies labelled with Alexa Fluor® 568-conjugated goat anti-rabbit Immunoglobulin G (IgG; cat. no. A-11034; dilution, 1:250; Invitrogen; Thermo Fisher Scientific, Inc.) and Alexa Fluor® 488-conjugated goat anti-mouse IgG (cat. no. A32727; dilution, 1:250; Invitrogen; Thermo Fisher Scientific, Inc.). Cell nuclei were stained with DAPI at room temperature for 20 min. Images were obtained using an E2000U confocal microscope (magnification, ×200 and ×400; Nikon Corporation, Tokyo, Japan) and were merged using Image Pro Plus (version 6.0; Media Cybernetics, Rockville, MD, USA).
MOVAS cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco's Modified Eagle's medium (DMEM) containing 10% FBS (cat. no. 10099-141; Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere at 37°C with 5% CO2. MOVAS cells were treated with various concentrations of Ang II (1, 10, 20, 50 and 100 nM) for 24 h. c-Jun small interfering RNA (siRNA), designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China), was transfected at a final concentration of 50 nM using Lipofectamine® 2000 (cat. no. 11668019; Invitrogen; Thermo Fisher Scientific, Inc.) for 24 or 48 h prior to subsequent experimentation, following the manufacturer's protocol. The c-Jun-si1 target sequence was 5′-GCCAACTCATGCTAACGCA-3′, the c-Jun-si2 target sequence was 5′-CAGCTTCCTGCCTTTGTAA-3′, and the c-Jun-si3 target sequence was 5′-GCGCATGAGGAACCGCATT-3′.
MOVAS cells (4×105 cells/well) were plated in 6-well glass slide chambers (Iwaki Glass, Tokyo, Japan) and treated with (or without) Ang II (20 nM) for 36 h. Cells were washed with PBS twice and fixed in 4% paraformaldehyde for 15 min at room temperature. Following washing with PBS, cells were blocked in fresh bovine serum albumin (BSA; cat. no. AR1006; Boster Biological Technology) buffer (0.5% Triton X-100, 2% BSA, and 0.1% Tween 20 dissolved in PBS) at room temperature for 1 h, and incubated with the primary antibodies anti-c-Jun/AP-1, anti-Chop (cat. no. GB11024; dilution, 1:100; Servicebio, Inc., Boston, MA, USA) and anti-α-SMA, overnight at 4°C. Cells were treated with secondary antibodies (goat anti-mouse IgG) and images were obtained as aforementioned.
Apoptotic cells characterized by DNA fragmentation in aortic tissues and MOVAS cells were determined by a TUNEL assay using an In Situ Cell Death Detection kit (cat. no. 11684817910; Roche Applied Science, Penzberg, Germany). First, aortic tissues were fixed as aforementioned, dehydrated, embedded, sectioned (5-µm thickness) and incubated with 0.9% NaCl for 10 min at room temperature. Sections were washed twice with PBS, and then incubated with biotinylated nucleotides and terminal deoxynucleotidyl transferase at 37°C for 1 h, followed by incubation with 50 µl TUNEL reaction mixture for 1 h at 37°C in a dark and humidified room. Sections were then stained with DAPI (5 µg/ml) at room temperature for 20 min. Following four washes in PBS, samples were mounted using anti-fade mounting medium (cat. no. IH0252; Beijing Leagene Biotech Co., Ltd., Beijing, China) and analyzed under a E2000U confocal microscope (4–7 fields were randomly selected; magnifications, ×200 and 400). The staining protocol of MOVAS cells was similar. Light green indicated normal DNA (TUNEL-) and bright green indicated damaged DNA (TUNEL+).
MOVAS cells were seeded in 96-well plates (5,000 cells/well), and cell proliferation was monitored every 24 h (24, 48 and 72 h) following treatment with Ang II using the CCK-8 assay (cat. no. CK04; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Following the manufacturer's protocol, the absorbance was read at 450 nm.
For the monolayer wound healing assay, MOVAS cells (6×105 cells/well) were seeded in 6-well plates, and a scratch was introduced on the cell layer after 24 h. A total of three separate wounds were scratched using a 200 µl pipette tip, and floating cells and cell debris were washed away with PBS. Fresh medium with 10% FBS was added, along with 20 nM Ang II for the experimental group, with continued cultured at 37°C. The wound was imaged at 0 and 24 h, under a transmission microscope (magnification, ×100), and the gap distances were measured.
Mouse aortas were used for protein extraction with radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology, Haimen, China), following centrifugation at 12,000 × g for 20 min at 4°C. Protein concentrations were determined using the bicinchoninic acid assay kit (cat. no. WLA004a; Wanleibio, Co., Ltd., Shanghai, China), according to the manufacturer's protocol. For western blotting, cell lysates (~35 µg per lane) were resolved via 10% (wt/vol) SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Merck KGaA). The membranes were blocked with 5% non-fat dry milk in TBS-Tween 20 for 1.5 h at room temperature, and incubated overnight at 4°C with primary antibodies against c-Jun/Ap-1 (cat. no. 711202; dilution, 1:200; Invitrogen; Thermo Fisher Scientific, Inc.), Chop (cat. no. mAb 2895; dilution 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), GAPDH (cat. no. A00227-1; dilution 1:100; Boster Biological Technology), and β-actin (cat. no. BM0627; dilution 1:100; Boster Biological Technology). The membranes were washed four times, and incubated with the horseradish peroxidase-conjugated secondary antibody (cat. no. 111-035-003; dilution 1:8,000; Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) in blocking buffer for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). ImageJ 1.42q (National Institutes of Health, Bethesda, MD, USA) was used for densitometry analyses.
TRIzol® (cat. no. 10296028; Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from MOVAS cells following treatment. RNA samples were reverse-transcribed into cDNA with different primers using the First Strand Synthesis kit (RR036A; Takara Biotechnology Co., Ltd., Dalian, China), following the manufacturer's protocol. The RT reaction was conducted at 37°C for 15 min and then at 85°C for 5 sec. cDNA was then stored at −20°C until use. qPCR was performed using SYBR® Premix Ex Taq (RR820A; Takara Biotechnology Co., Ltd.) and an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed as follows: 95°C for 30 sec, and then 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Relative gene expression was determined using the 2−∆∆Cq method (
The mouse Ddit3 promoter region sequence (−1.09 kb to 0 kb) was downloaded from the UCSC Genome Browser (
A ChIP kit was purchased from Beyotime Institute of Biotechnology (cat. no. P2078), and ChIP assay was conducted following the manufacturer's protocol. MOVAS cells were cross-linked (40% methanol solution, 37°C for 15 min) and then sonicated (0°C, 50% power with 4 cycles of 5 sec on, 5 sec off) when they reached 90% confluency in 10 cm cell culture dishes, followed by IP with a polyclonal anti-c-Jun/Ap-1 antibody (cat. no. 711202; Invitrogen; Thermo Fisher Scientific, Inc.). Normal IgG (cat. no. ab172730; Abcam, Cambridge, UK) was used as a negative control. The supernatant was used as an input control. Precipitated DNA was amplified by PCR using Ddit3-specific primers (forward, 5′-CTGAGTGGCGGATGTAAGGG-3′; reverse, 5′-GGTCCAGGAGCCTACCAATC-3′). PCR products (74 bp) were analyzed by 2% agarose gel (cat. no. 5261; Takara Biotechnology Co., Ltd.) electrophoresis and visualized using GoldView™ (cat. no. G8140; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) under an ultraviolet light.
All results are presented as the mean ± standard error of the mean from three independent experiments. Statistical differences were evaluated by one-way analysis of variance followed by a Tukey's comparison test. Statistical significance was evaluated using GraphPad Prism (version 5.0; GraphPad Software Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
After 4 weeks of Ang II treatment, AAA developed in ApoE−/− mice but not in saline-infused ApoE−/− mice, which exhibited normal aortas (
To investigate the role of Ang II in the cell proliferation and migration of MOVAS cells, CCK-8 and monolayer wound healing assays were performed. The results demonstrated that Ang II did not affect MOVAS cell proliferation (
To investigate the expression of c-Jun during Ang II-induced AAA formation, immunofluorescence was used. c-Jun levels were observed to be higher in Ang II-induced AAA compared with normal aortas (
Based on the aforementioned results, it was hypothesized that there may be underlying connections between c-Jun/Ap-1 and Ddit3. AP-1 is a transcription factor consisting of a dimer of c-Jun and c-Fos. However, whether Ddit3 is activated by Ap-1 is unknown. Following this line of thought, c-Jun was downregulated using c-Jun-targeted siRNAs and the expression of Chop was assessed. RT-qPCR revealed that c-Jun transcript levels were reduced by >60% at 24 h post-transfection with c-Jun-si2 and c-Jun-si3 [P<0.001 vs. negative control (NC)-si], but c-Jun-si1 had no effect (P>0.05 vs. NC-si). GAPDH was used as an internal control (
To further evaluate the aforementioned hypothesis, the promoter sequence of mouse Ddit3 (encoding Chop) was evaluated using online databases (−1.09 kb to 0 kb). The sequence analysis suggested five potential c-Jun/Ap-1 binding sites in the Ddit3 promoter region (
Previous evidence suggests that Ang II serves important roles in cardiovascular homeostasis by directly regulating ER signaling and upregulating Chop expression (
A number of AAA induction methods in animal models has been developed, and three approaches are frequently used by researchers, including the porcine pancreatic elastase model (
c-Jun/AP-1 and Chop were also observed to be upregulated in Ang II-induced AAA and Ang II-treated MOVAS cells. Additionally, the present results demonstrated that the increase in c-Jun and Chop expression levels in response to Ang II was dose-dependent, with c-Jun and Chop expression levels decreasing at higher doses (100 nM). A possible interpretation is that Ang II may induce ER stress, intracellular reactive oxygen species generation and inflammation, leading to cell apoptosis. A previous study demonstrated that VSMC cell apoptosis was directly dependent on Ang II concentration, and that 50 nM Ang II may induce 25–30% cell apoptosis; 100 nM Ang II led to cell death without activation of an apoptotic pathway (
The present work had certain limitations. Firstly, the focus of the study was to determine whether c-Jun/AP-1 may mediate Chop expression. In the
In conclusion, the present study demonstrated that c-Jun/AP-1 was overexpressed in an Ang II-induced AAA model and in Ang II-treated MOVAS cells, and that it mediated the expression of Chop. Therefore, c-Jun/AP-1 may be a novel target for AAA therapy.
Not applicable.
This study was funded by research grants from The National Natural Science Foundation of China (grant no. 81370417) and Hubei Natural Science Foundation Project (grant no. 2018CFB465).
All data generated or analyzed during the present study are included in this published article.
JY and MZ were involved in the conception and supervision of the project. JY and DS were involved in the design of the study. DS performed the experiments. DS, YL and JQ analyzed the results. DS and SM performed and analyzed the CCK-8 and migration assays. DS, YL and JQ prepared the paper. All the authors read and approved the final manuscript.
All animal experiments were performed in accordance with the Animal Research Reporting of
Not applicable.
The authors declare that they have no competing interests.
abdominal aortic aneurysm
angiotensin II
activator protein-1
C/EBP homologous protein
apolipoprotein E-deficient
chromatin immunoprecipitation
AAA is induced by Ang II in ApoE−/− mice. (A) Morphology of Ang II-induced AAA and normal aortas (control) in ApoE−/− mice; white arrows indicate a typical AAA. (B) Aorta cross-sections stained with hematoxylin and eosin. (C) Representative immunofluorescence and TUNEL staining for the detection of DNA fragmentation in Ang II-induced AAA and saline-treated aortas (magnification, ×200). (D) TUNEL assay for detecting cell apoptosis in MOVAS cells treated (or not treated, saline) with Ang II by fluorescence microscopy (magnification, ×400). Light green indicates normal DNA (TUNEL-) and bright green indicates damaged DNA (TUNEL+). **P<0.01 vs. respective Sham. AAA, abdominal aortic aneurysm; Ang II, angiotensin II; ApoE−/−, apolipoprotein E-deficient; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; α-SMA, α-smooth muscle actin.
Ang II induces cell migration but does not affect cell proliferation. (A) CCK-8 assay growth curves revealed that Ang II had no effect on the proliferation of MOVAS cells (cells were treated with Ang II 20 nM, or were untreated, for 0, 24, 48 and 72 h. (B) The wound-healing assay demonstrated that the migration ability of MOVAS cells was increased following treatment with 20 nM of Ang II. Scale bar, 100 mm. **P<0.01 vs. Sham; n≥3. Ang II, angiotensin II; CCK-8, Cell Counting Kit-8; OD450, optical density at 450 nm; ns, non-significant.
Ap-1 and Chop are induced by Ang II
Downregulation of c-Jun/Ap-1 by c-Jun-siRNA suppresses Chop expression. (A) mRNA levels of c-Jun were assessed by RT-qPCR in MOVAS cells transfected with siRNAs for 24 h; GAPDH was used as an internal control (n≥3). (B) Western blotting demonstrated the Ap-1 levels in MOVAS cells transfected with siRNAs for 48 h; GAPDH was used as an internal control (n=3). MOVAS cells were transfected with siRNAs for 24 h and the transfected cells were treated with Ang II (20 nM) for 24 h. (C) The mRNA expression level of Ddit3 were detected by RT-qPCR; GAPDH was used as an internal control (n≥3). (D) Protein levels were detected by western blotting with anti-c-Jun, anti-Chop, and anti-β-actin (internal control) antibodies (n=3). All results are presented as the mean ± standard error of the mean. #P<0.05; *P<0.05, **P<0.01, ***P<0.001 vs. NC-si. Ap-1, activator protein 1; Chop, C/EBP homologous protein; RT-qPCR, reverse transcription quantitative-polymerase chain reaction; siRNA, small interfering RNA; Ang II, angiotensin II; NC-si, negative control siRNA; Ddit3, DNA damage-inducible transcript 3 protein.
c-Jun/Ap-1 binds to the mouse Ddit3 promoter region in MOVAS cells. (A) Sequence of the mouse Ddit3 promoter region (−1090 to 0) and sketch of putative AP-1 binding sites. (B) Binding of Ap-1 to the mouse Ddit3 promoter was detected via ChIP-PCR using Ddit3 promoter-specific primers. An anti-Ap-1 antibody was used for IP in ChIP assays, and anti-rabbit IgG was used for control experiments. DNA fragments were obtained after cell cross-linking and sonication. Ap-1, activator protein 1; Ddit3, DNA damage-inducible transcript 3 protein; ChIP, chromatin immunoprecipitation; PCR, polymerase chain reaction; IgG, immunoglobulin G; TSS, transcription start sequence.