Activation of activator protein 2 alpha by aspirin alleviates atherosclerotic plaque growth and instability in vivo

Aims Aspirin has been used for the secondary prevention and treatment of cardiovascular disease for several decades. We investigated the roles of transcriptional factor activator protein 2α (AP-2α) in the beneficial effects of aspirin in the growth and vulnerability of atherosclerotic plaque. Methods and Results In mice deficient of apolipoprotein E (Apoe-/-), aspirin (20, 50 mg/kg/day) suppressed the progression of atherosclerosis in aortic roots and increased the plaque stability in carotid atherosclerotic plaques induced by collar-placement. In vivo lentivirus-mediated RNA interference of AP-2α reversed the inhibitory effects of aspirin on atherosclerosis in Apoe-/- mice. Mechanically, aspirin increased AP-2α phosphorylation and its activity, upregulated IkBα mRNA and protein levels, and reduced oxidative stress in cultured vascular smooth muscle cells. Furthermore, deficiency of AP-2α completely abolished aspirin-induced upregulation of IkBα levels and inhibition of oxidative stress in Apoe-/- mice. Clinically, conventional doses of aspirin increased AP-2α phosphorylation and IkBα protein expression in humans subjects. Conclusion Aspirin activates AP-2α to upregulate IkBα gene expression, resulting in attenuations of plaque development and instability in atherosclerosis.

2 around the left common carotid artery. In brief, a constrictive silastic tube (0.30-mm inner diameter, 0.50-mm outer diameter, and 2.5-mm long; Shandong Key Laboratory of Medical Polymer Materials, Jinan, China) was placed around the left common carotid artery near its bifurcation in male Apoe −/− mice at age of 8-12 weeks. Mice were then fed with a high fat diet (0.25% cholesterol and 15% cocoa butter) plus or minus aspirin administration (5,20, 50 mg/kg/day) in drinking water for 8 weeks. At the end of experiment, all mice were sacrificed under anesthesia. The thoracic and abdominal aortas were collected for histological analysis of atherosclerotic lesion growth. The left common carotid arteries were collected for pathological analysis of atherosclerotic plaque stability.
In the second part of the animal study, as shown in Supplementary Figure   S2A, male Apoe -/mice at age of 8-12 weeks were infected with lentivirus containing negative control shRNA or AP-2α shRNA (once 4 weeks) via tail vein as described previously with light modifications 1 and received a high fat diet (0.25% cholesterol and 15% cocoa butter). 4 weeks later, aspirin (50 mg/kg/day per mouse) was added into drinking water and maintained for 8 weeks. At the end of experiment, mice were euthanized with an intraperitoneal injection of 0.8% pentobarbital sodium (60 mg/kg), followed by cervical dislocation. The aortas were collected for histological and molecular biological analysis.
In the third part of in vivo study, as shown in Supplementary Figure S2C, male Apoe -/mice at age of 8-12 weeks had left carotid collar placement plus local lentivirus infection as described previously 2 . After surgery, mice were fed a high fat diet (0.25% cholesterol and 15% cocoa butter). 4 weeks later, mice received aspirin administration in drinking water (50 mg/kg/day) and injection of lentivirus (once every 4 weeks) and kept for 8 weeks. The left common carotid arteries were collected for pathological and molecular biological analysis.

Generation of shRNA construct and lentivirus production
Based on the protocol from Signaling Gateway, the shRNA cassette containing target sequence of AP-2α (GGAGAGCGAAGTCTAAGAATG) was designed.
The cassette was subcloned into pEN-hH1c vector as described previously 3 .
The pEN-hH1c vector containing the AP-2α shRNA cassette was combined 3 with an attR-containing vector pDSL-hpUP in an LR recombination reaction.
The recombinant constructs pDSL-hpUP-AP-2α-shRNA were confirmed by DNA sequence analysis. The sequence of negative control shRNA is TTCTCCGAACGTGTCACGT. The lentivirus was produced by transiently transfecting HEK293T cells using SuperFect transfection reagent (Qiagen, USA) with three packing plasmid system (pGag/Pol, pRev, and pVSV-G).The virus-containing supernatant was collected 72 hours after transfection, and filtered through 0.45 mm filters (Millipore, USA), and stored at -80 °C. The titer of the viral vectors was determined by TCID50 (Tissue culture infective dose) method.

Cell cultures
Human vascular smooth muscle cells (VSMCs) from ATCC were grown in basal medium (Clonetics Inc. Walkersville, MD) supplemented with 2% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). In all experiments, cells were between passages 3 and 8. All cells were incubated at 37°C in a humidified atmosphere of 5% CO 2 and 95% air. Cells were grown to 70-80% confluency before being treated with different agents.

Generation and infection of adenoviral infection to cells
To generate adenoviral vector containing Ad-S219A-AP-2α cDNA, we subcloned a human cDNA encoding full-length of AP-2α, which contained a substituted amino acid of serine 219 to alanine (S219A), into a shuttle vector (pShuttle CMV [cytomegalovirus]) as described previously 4 . VSMCs were infected with adenovirus overnight in medium supplemented with 2% FBS. The cells were then washed and incubated in fresh medium for an additional 24 hours before experimentation. These conditions typically produced an infection efficiency of at least 80%.

Western blot analysis
Cell lysates or tissue homogenates were subjected to western blot analysis, as described previously 5 . The protein content was assayed by BCA protein assay reagent (Pierce, USA). Protein of 20 µg was loaded to SDS-PAGE and then transferred to membrane. Membrane was incubated with a 1:1000 dilution of primary antibody, followed by a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized by ECL (GE Healthcare). The intensity (area × density) of the individual bands on Western blots was measured by densitometry (model GS-700, Imaging Densitometer; Bio-Rad). The background was subtracted from the calculated area. We used control as 100%.

Detection of ROS
ROS productions in cultured cells were assayed by measuring the DHE fluorescence by HPLC with minor modification 6

DNA-binding activities
Subcellular fractions were prepared by using NE-PER Nuclear and Cytoplasmic Extract kit (Cat78833) from PIERCE. EMSA were performed as described in a previous study 7 . AP-2α kit (AY1002) is from Panomics Company.

ChIP assay for transcriptional factor and gene promoter binding
ChIP assays were performed by using a ChIP-IT kit (Upstate, 17-295), according to the manufacturer's protocol. 1×10 6 cells were seeded on a 10 cm dish. Proteins were cross-linked to DNA by adding formaldehyde directly to culture medium at a final concentration of 1% and incubating for 10 min at 37ºC.
The cells were harvested in SDS lysis buffer and added protease inhibitors.
Cell lysates were sonicated to shear DNA to lengths between 200 and 1000 bp.

Immunohistochemistry (IHC)
The thoracic aorta was fixed in 4% paraformaldehyde overnight, and then

Atherosclerotic lesion analysis
After being fed the Western diet for 8 weeks, the mice were fasted for 14 h and then were anesthetized and euthanized. The heart and aortic tissue were removed from the ascending aorta to the ileal bifurcation and placed in 4% paraformaldehyde for 16 h. After fixation, the adventitia was thoroughly cleaned under a dissecting microscope. For analyzing the lesion area in the aortic root, the heart was dissected from the aorta, embedded in Polyfreeze tissue freezing medium (Polysciences, Inc) and sectioned (5 µm thickness).
Four consecutive sections were collected from each mouse and stained with 6 Oil Red O for neutral lipids, and counterstained with hematoxylin to visualize the nuclei. Plaques were captured under the Olympus microscope connected to a QImaging Retiga CCD camera. The aortic lesion size of each animal was obtained by the averaging of lesion areas in four sections from the same mouse. For analyzing the lesion area in the aortic arch, the intimal surface was exposed by a longitudinal cut from the ascending arch to 5 mm distal of the left subclavian artery to allow the lumen of the aortic arch to be laid flat. The aorta was rinsed for 5 min in 75% ethanol, stained with 0.5% Sudan IV in 35% ethanol and 50% acetone for 15 min, destained in 75% ethanol for 5 min, then rinsed with distilled water. Digital images of the aorta were captured under a stereomicroscopy, and the lesion area was quantified from the aortic arch to 5 mm distal of the left subclavian artery using Alpha Ease FC software (version 4.0 Alpha Innotech). The plaque vulnerable index was calculated according to the ratio of area I (Oil Red + + CD68 + ) to area II (α-SMA + + Collagen + ) as described previously 8 .

Measurement F 2 -isoprostanes in urine and blood
The concentration of iPF 2 α-VI in urine or blood was determined by LC-MS/MS as previously described 9 . In brief, 0.1 ml of 10 ng/ml deuterated internal standard (8iPF2α-d4; Cayman chemical, Ann Arbor, MI, USA) was added to 1 ml urine or blood. The sample was then subjected to solid phase extraction (Oasis HLB, Waters, Milford, MA, USA). The eluate was taken to dryness under a stream of nitrogen at room temperature, and afterwards redissolved in 100 μl 10% acetonitrile of which, 40 μl was injected on a reverse-phase XTerra MS C18 column (Waters, Milford, MA, USA; 3.5 μm, 2.1X100 mm). Urinary F2-isoprostanes were quantified using a Quattro Micro (Waters) mass spectrometer. To calculate the iPF 2 α-VI concentration, the analyte to internal standard peak area ratio was compared with a standard curve from 2 to 16 ng/ml iPF 2 α-VI (Cayman chemical, Ann Arbor, MI, USA). The intra-run coefficient of variation (CV) was 4.8% and the inter-run CV was 10.1%.

Statistical analysis
All quantitative data are reported as mean ± SEM. and were analyzed using a two-way ANOVA. Bonferroni corrections were applied to multiple comparisons and P<0.05 was considered significant.