2.1 Isolation as well as culture of vascular smooth muscle cells (VSMCs)

The primary mouse VSMCs were abstracted from the media of mouse aortas of ApoE^−/− mice in Dulbecco's modified eagle medium (DMEM, GIBCO) complemented with 20% foetal bovine serum (FBS, GIBCO), 100 U/mL of penicillin plus 100 μg/mL of streptomycin at 37°C in 5% CO₂. Briefly, 6-week-old ApoE^−/− male mice were used to obtain the aortas and the vascular adventitia was removed under a microscope. After washing the aortas with PBS along with 100 U/mL of penicillin as well as 100 μg/mL of streptomycin three times, the aortas were cut into about 1 mm length explants by a sterile Venus. The explants were individually plated into a 6-cm culture dish and cultured in DMEM containing 20% FBS for 7 days before the next change of medium. The acquired cells reserved SMC features (purity >90%). Cells at passages 3-9 were used for experiments.

2.2 Preparation of lentiviral vectors and target screening of siRNA

The siRNA negative control (si-NC) was a scramble siRNA sequence with unidentified homology to mammal genes (GeneChem). The siRNA sequence of the mouse NONO gene was documented in Supplementary material10. The siRNA sequence was also described and confirmed previously as follows: CCCACCAACAACTGAACGTTTCTCGAGAAACGTTCAGTTGTTGGTGGG.

The replication-incompetent lentivirus (LV) was created by siRNA expression vector with pHelper 1.0 as well as pHelper 2.0 into 293T cells. At 48 hours after transfection, viral supernatant was harvested and filtered via a 0.45 cellulose acetate filter. The titre of the viral vectors was detected by fluorescence-activated cell sorting analysis of GFP-positive 293T cells (GeneChem).

sh-NONO-LV was transduced into VSMCs (5 × l0⁵ cells in 12-well plates) at a multiplicity of infection (MOI) of 100. At day 3 after transduction, cells were collected for Western blot in order to detect the efficiency and screen the most effective sh-RNA of NONO. Besides, sh-NONO was transduced into VSMCs for different treatments.

2.3 Animals

All animal experimental protocols were permitted by the Ethics Committee and the Scientific Investigation Board of Shandong University Qilu Hospital according to the Animal Management Rules of the Chinese Ministry of Health. 8-week-old apolipoprotein E-knockout (ApoE^−/−) male mice were bought from Beijing Weitonglihua Animal Experimental Center. All animals were kept on a 12-hour/12-hour light-dark cycle at 22°C room temperature with the high-fat food (0.25% cholesterol plus 15% cocoa butter) and water freely available for 8 weeks.

During the first part of the in vivo experimentation, 20 ApoE^−/− male mice receiving high-fat diet were arbitrarily distributed into the control group (n = 10) as well as model group (n = 10). Mice in model group were treated with continuous subcutaneous infusion of Ang II (1.44 mg/kg per day, Lintai Biological Technology Company, Xi'an, China) by an osmotic pump (Alzet model 2004, Alza Corp.) for 28 days3 in order to examine the expression of NONO on Ang II-influenced AAA.

During the second part, 20 ApoE^−/− male mice receiving high-fat diet were used. These mice were transfected with sh-NC (2 × 10⁷ TU per mice) from tail vein injection. After 4 weeks, all mice were operated with osmotic pumps to receive continuous subcutaneous infusion of Ang II. We sacrificed the animals prior to the transfection (n = 5), 4 weeks following the transfection (n = 5), 6 weeks following the transfection (n = 5), as well as 8 weeks following the transfection (n = 5) to inspect the transfection competence of lentivirus by detecting GFP fluorescence.

In the third part, 100 ApoE^−/− male mice receiving high-fat diet were arbitrarily separated into two groups: one was the control group without Ang II infusion (n = 25), and another was the Ang II group. The control group received no lentivirus injection and no infusion of Ang II. Mice treated with Ang II were further arbitrarily distributed into three groups (n = 25/group): No treatment group (NT group) received infusion of Ang II after 4 weeks of 0.9% saline injection; Sh-NC group received infusion of Ang II following 4 weeks of sh-NC (2 × 10⁷ TU per mice) injection, and sh-NONO group received infusion of Ang II following 4 weeks of sh-NONO (2 × 10⁷ TU per mice) injection.

Ang II was constantly infused into mice subcutaneously through an osmotic pump for 28 days. The aortic tissues were removed from mice with euthanasia after 28-day infusion. After 8 weeks, all ApoE^−/− mice were sacrificed to obtain blood from the left ventricle and the intact aortas.

2.4 Bodyweight and serum lipid profile

The bodyweight of animals was measured at the beginning and at the end of the experiment by an electronic balance (Shimadzu Corp.). Blood samples were acquired through cardiac puncture prior to euthanasia. The serum levels were measured by an enzymatic assay, containing total cholesterol, triglycerides, low-density lipoprotein cholesterol as well as high-density lipoprotein cholesterol.

2.5 Measurement of blood pressure (BP)

Systolic blood pressure (SBP) was obtained using a tail-cuff method with a photoelectric device (Natsume, Japan) before Ang II infusion and before anaesthesia in all mice following the beginning of the experimentation. Blood pressure was stated as a mean of three successive recordings.

2.6 AAA quantification

Aortas were extracted and fixed in 4% paraformaldehyde overnight. The adventitia was detached under the microscope. The highest diameter of the abdominal aorta was obtained using Image-Pro Plus 6.0 software for aneurysm quantification. Abdominal aortic aneurysm was defined as the 150% diameter increase criterion. Besides, any dissection leading to intramural haematoma (even if the actual dilatation is only 110%) should be counted in the AAA mouse model.15 The severity of AAA was classified as described previously: None: no aneurysm, Type I (lumen dilatation without intraluminal thrombus), Type II (lumen dilatation with thrombus), Type III (conspicuous bulbous form of Type II with thrombus) and Type IV (multiple aneurysms with thrombus, with some aneurysms overlapping).16 Two autonomous researchers were in charge for the measurements.

2.7 Tissue preparation and histopathological analysis

Aortic fragments from the aortic arch to the renal arteries were detached, fixed in 4% paraformaldehyde overnight and then paraffin-embedded. 10 tissue sections, 5 μm thick, were derived from the AAA portion with the largest diameter in each mouse, which were used for histological as well as morphological investigation.

Aortic fragments of all groups were used for various staining procedures. Haematoxylin and eosin (H&E) staining was implemented to assess morphology of AAA. Verhoff staining was done to visualize the elastic fibre integrity of the abdominal aorta through a Verhoff-Van Gieson staining kit (Abcam). Masson staining and Sirius Red staining were implemented to detect the aortic collagen content.

Sections of aneurysm were stained for immunohistochemical analysis of macrophages (CD68, abcam, ab125212, 1:100), smooth muscle cells (α-SM actin, α-SMA, abcam, ab7817, 1:200), collagen I (abcam, ab34710, 1:200), collagen III (abcam, ab7778, 1:200), P4Hα1 (abcam, ab59497, 1:200), MMP2 (abcam, ab37150, 1:200), MMP9 (abclonal, A2095, 1:200), IL-1β (abcam, ab9722, 1:200), MCP-1 (abcam, ab9669, 1:200), IL-6 (abcam, ab7737, 1:200) as well as TNF-α (abcam, ab1793, 1:400). Following the incubation with the suitable horseradish peroxidase-conjugated secondary antibodies, fragments were incubated with 3′, 3′-diaminobenzidine, counterstained, then stained with haematoxylin, desiccated with gradient alcohol and protected with coverslips. Sections reacting with non-immune IgG as well as secondary antibodies were considered as negative controls by Image J software (NIH) was used to calculate the ratio of positive staining region to total plaque area.

2.8 Cell treatment

Vascular smooth muscle cells were cultured in DMEM complemented with 20% FBS with antibiotics. First, VSMCs were enthused by Ang II (MCE, 1 × 10⁻⁷ mol/L) for the Ang II stimulation study. Cells were harvested for measurement of NONO expression levels. Second, VSMCs cultured in 6-well plates were transfected by lentivirus (MOI = 100, per group) comprising sh-NC or sh-NONO overnight. The Ang Il group was administered physiological saline of the similar volume. Three days following the transduction, cells were treated with 1 × 10⁻⁷ mol/L Ang II for 24 hours and then collected for additional investigation. All experimentations were done thrice plus the mean values were obtained.

2.9 Western blot technique

Total protein was extracted from the aortas of ApoE^−/− mice or VSMCs. The aortic segments from the aortic arch to the renal arteries were collected and used for Western blotting. The protein concentrations were quantified by a Protein Assay Kit (Thermo fisher). Equivalent quantities of protein were separated by 10% polyacrylamide gel electrophoresis and transferred to 0.22 μm polyvinylidene fluoride (PVDF) membranes. Following the blocking for 1 hour at room temperature (RT) with 5% skim milk, overnight incubations of membranes were done at 4°C with appropriate primary antibodies. Mouse anti-NONO (Santa Cruz, sc-166702x, 1:1000), rabbit anti-collagen I (Cell Signaling Technology, 84336S, 1:1000), rabbit anti-collagen III (abcam, ab7778, 1:1000), goat anti-P4Hα1 (abcam, ab59497, 1:1000), rabbit anti-MMP2 (abcam, ab37150, 1:1000), rabbit anti-MMP9 (abclonal, A2095, 1:1000), rabbit anti-IL-1β (abcam, ab9722, 1:1000), rabbit anti-monocyte chemotactic protein 1 (MCP-1) (abcam, ab9669, 1:1000), rabbit anti-TNF-α (abcam, ab1793, 1:1000). Besides, rabbit anti-Phospho-NF-κB p65 (p-p65) (abcam, ab76302, 1:1000) and rabbit anti-NF-κB p65 (Cell Signaling Technology, 8424, 1:1000) were used. Following washing, membranes were incubated with the analogous horseradish peroxidase-conjugated secondary antibody (Jackson) for 1 hour at RT, then washed and detected by chemiluminescent HRP substrate (Millipore). GAPDH immunoblot analysis was used to verify equal sample loading.

2.10 Co-immunoprecipitation

Cell extracts used for co-immunoprecipitation were prepared from VSMCs, and co-immunoprecipitation analysis was carried out as described previously.5 Briefly, VSMCs were cultured in a 150 mm² culture bottle. Cells with 80%-90% confluency were cultured in serum-free medium for 24 hours for synchronization. Next, cells were stimulated with or without 1 × 10⁻⁷ mol/L Ang II for 1 hour and lysed in 1 mL cold RIPA buffer containing 10 μL PMSF (100 mM). Specific antibodies (anti-NONO and anti- NF-κB p65) or pre-immune IgGs were incubated with cell lysates overnight before getting immersed by Protein A/G-plus Agarose beads (Santa Cruz, USA). Precipitating immune complex was unconfined by boiling with 1X SDS electrophoresis sample buffer. Bound proteins were identified by Western blot technique.

2.11 Immunofluorescence

Vascular smooth muscle cells were cultured, transfected with si-NONO and stimulated with Ang II as explained earlier. Cells were fixed using 4% paraformaldehyde for 20 minutes and permeabilized in PBS with 0.1% Triton X-100. Following the blocking with goat serum for 30 minutes at RT, samples were incubated with rabbit anti-NF-κB p65 (Cell Signaling Technology, 8424, 1:100) and mouse anti-NONO (Santa Cruz, sc-166702x, 1:100) antibodies overnight at 4°C. Alexa 488-conjugated goat anti-mouse IgG and Alexa 594-conjugated goat anti-rabbit IgG (Invitrogen) were used as the secondary antibodies. IgG and secondary antibodies were considered as negative controls. A droplet of Prolong Gold anti-fade reagent with DAPI (Vector Laboratories) was added to fix the coverslip. Images were captured via laser scanning confocal microscopy (LSM 710, Zeiss).

2.12 Statistical analysis

SPSS v19.0 (SPSS Inc) was used for data analysis. Data are presented as mean ± SD. For the comparison between two groups, an independent samples t test was used. One-way ANOVA analysis was used for multiple comparisons when the homogeneity of variance assumptions is fulfilled; otherwise, the equivalent non-parametric test was used. Chi-square test was used for multiple comparisons. The status of mice was observed and Kaplan-Meier curves were plotted during the experiment. P < .05 signified statistical significance.

3.1 NONO expression in AAA as well as Ang II-induced VSMCs

To explore the incorporation of NONO in AAA, we investigated the manifestation level of NONO in AAA tissues of ApoE^−/− mice as described in the first part of the flow chart (Figure S1). We found that the expression of NONO was suggestively augmented in AAA tissues of ApoE^−/− mice compare to the control group (1.14 ± 0.19 vs 1.53 ± 0.23, P < .05, N = 6; Figure 1A,B), indicating that NONO may have involvement in the advancement of AAA. We also investigated NONO protein manifestation levels in Ang II-stimulated VSMCs and found that Ang II markedly increased NONO expression level (1.16 ± 0.15 vs 1.80 ± 0.28, P < .05, N = 3; Figure 1C,D). Furthermore, we also investigated the expression of NONO in SMC, endothelial cells or macrophages by double immunofluorescence in AAA tissues. The positive cells of these cells were 2033 ± 234.9, 1230 ± 364.1 and 979.7 ± 240.4 respectively. We found that all the above cells expressed NONO protein, and the co-expression rate was 13.36 ± 1.29, 12.92 ± 2.78 and 11.18 ± 2.35. SMCs were in the majority in AAA tissues (Figure 1E-G). Our data demonstrated that NONO was expressed in the normal aortas; importantly, the expression levels of NONO were augmented in both AAA and Ang II-induced VSMCs which suggested that NONO might response to Ang II and participate in the expansion of AAA.

3.2 Efficiency of lentiviral transfection in vivo and in vitro

The lentivirus with GFP reporter gene was transfected into AAA tissues by tail intravenous injection, and then, GFP fluorescence was examined in AAA tissues at 4, 6 and 8 weeks following the transfection in the second part of the in vivo experimentation (Figure S1). Following the transfection for 4 weeks, GFP fluorescence was significantly detected in AAA (Figure S2A) and both 6 and 8 weeks after transfection, fluorescence intensity of GFP was still visible (Figure S2A).

Abdominal aortic aneurysm tissues were transfected with sh-NONO-LV and the transfection conferred about 50% reduction in the NONO protein expression (1.18 ± 0.10 vs 0.57 ± 0.03, P < .01; Figure S2B,C). Similar results were also observed in VSMCs transfected with or without sh-NONO-LV (1.25 ± 0.12 vs 0.58 ± 0.03, P < .01; Figure S2D,E).

3.3 Serum lipid levels and blood pressure

Serum lipid levels were detected, and no difference was found among the four groups in the third part of the in vivo experimentation (Figure S1 and Table S1). Systolic blood pressure (SBP) was suggestively augmented at 8 weeks following the transfection in the Ang II-infused ApoE^−/− mice compare to their baseline levels or in control group. However, NONO knockdown had no effects on SBP (Table S1).

3.4 Effect of NONO down-regulation on the occurrence of AAA in Ang II–infused ApoE^−/− Mice

In ApoE^−/− mice, in the third part of in vivo experimentations (Figure S1), the incidence of AAA was 80%, 84% and 48% in the no treatment, sh-NC and sh-NONO groups, respectively, compared with that in the control group which had no AAA (Figure 2A-D and Figure S3). Based on the severity classification method, we observed that Ang II induced more type III and IV forms in no treatment and sh-NC groups, whereas Ang II induced more type I and II forms in sh-NONO group (Figure 2C). Thus, NONO knockdown suggestively reduced the occurrence of AAA in the Ang II-infused ApoE^−/− mice. Moreover, the greatest abdominal aortic diameter was suggestively augmented in Ang II-infused ApoE^−/− mice compare to the control group (Figure 2E). NONO knockdown obviously reduced the diameter in the Ang II-infused ApoE^−/− mice, while there was not any noteworthy alteration in the diameter between the NT and sh-NC groups (Figure 2E). Kaplan-Meier curves showed that Ang II infusion lea to the decreased percent survival in ApoE^−/− mice (Figure 2F). Ang II increased death rate due to aneurysm rupture during AAA formation compared with the control group (P < .05), whereas NONO down-regulation reversed both high mortality (8%, 36%, 44% and 32%, P < .05 vs the control group, Figure 2G) and rupture rate compared with the control group (0, 24%, 28% and 12%, P < .05 vs the control group, #P < .05 vs no treatment group, &P < .05 vs Sh-NC group, Figure 2H).

3.5 Effect of NONO on Ang II-induced histological as well as morphological modifications in ApoE^−/− mouse aortas

H&E as well as Verhoff staining indicated that Ang II infusion induced positive remodelling during the pathological process of AAA in ApoE^−/− mice including hypertrophy and breakdown of the adventitia, destruction of the aortic media, and discontinuity of elastin fibres (Figures 2B and 3A). These pathological changes were largely attenuated in the sh-NONO group compared with those in the NT or sh-NC groups (Figures 2B and 3A). However, no difference was found in the morphology of the abdominal aorta between the NT and sh-NC group, which was characterized by aortic wall thickening, elastin fibre discontinuity and dilated abdominal aorta (Figure 3A).

Furthermore, contents of collagen (2.73 ± 0.50 vs 1.93 ± 0.47 vs 14.85 ± 1.35, P < .01) as well as SMCs (12.24 ± 1.44 vs 13.16 ± 2.10 vs 59.30 ± 8.13, P < .01) in the aortic wall were significantly augmented in the sh-NONO group, whereas the content of macrophages (37.88 ± 1.47 vs 36.80 ± 2.43 vs 10.12 ± 0.61, P < .01) was decreased in the sh-NONO compared with those in the NT or sh-NC group (Figure 3A-D). Additionally, there was not any substantial difference among the NT and sh-NC groups on contents of collagen, SMCs and macrophages (Figure 3A-D).

3.6 Effect of NONO on the collagen deposition and degradation in vivo as well as in vitro

To evaluate the impact of NONO on extracellular matrix deposition and degradation, we further investigated the manifestation levels of P4Hα1, type I collagen plus type III collagen, and the representative elastin degradation indicators MMP2 plus MMP9. In the in vivo experimentation, NONO knockdown significantly augmented collagen synthesis and deposition of Sirius Red staining (5.24 ± 0.77 vs 6.03 ± 0.89 vs 21.2 ± 0.37, P < .01), Collagen I (14.46 ± 1.40 vs 13.43 ± 1.04 vs 51.46 ± 4.14, P < .01), Collagen III (13.81 ± 1.87 vs 12.56 ± 0.90 vs 32.51 ± 1.504, P < .01) and P4Hα1 (4.92 ± 0.58 vs 6.10±0.0.61 vs 14.54 ± 1.27, P < .01), and decreased collagen degradation MMP2 (18.02 ± 1.18 vs 15.24±0.1.56 vs 6.36 ± 0.44, P < .01) and MMP9 (13.38 ± 1.54 vs 13.12 ± 1.08 vs 3.68 ± 0.85, P < .01) in aorta compared with those in the NT or sh-NC groups (Figure 4A-H). No substantial alteration was found between the NT and sh-NC groups on collagen deposition. In the in vitro experiment, knockdown of NONO also obviously augmented the manifestation levels of collagen I (1 vs 1.22 ± 0.12 vs 11.14 ± 1.42, P < .01), collagen III (1 vs 1.13 ± 0.18 vs 3.03 ± 0.19, P < .01) as well as P4Hα1 (1 vs 0.99 ± 0.12 vs 2.01 ± 0.18, P < .01) proteins, and reduced levels of MMP2 (1 vs 0.94 ± 0.03 vs 0.70 ± 0.05, P < .01) and MMP9 (1 vs 0.94 ± 0.10 vs 0.69 ± 0.07, P < .05) in VSMCs with Ang II stimulation (Figure 4I-K). Thus, silencing of NONO participated in promoting the collagen deposition and inhibiting the collagen degradation in AAA.

3.7 Effect of NONO on inflammation infiltration in vivo as well as in vitro

We also identified the impact of NONO on inflammation infiltration. Immunohistochemical staining analysis showed that NONO knockdown significantly reduced the manifestation levels of IL-1β (21.20 ± 1.10 vs 22.34 ± 1.57 vs 10.54 ± 0.88, P < .01), MCP-1 (15.32 ± 1.46 vs 13.96 ± 0.84 vs 8.59 ± 0.42, P < .01), IL-6 (14.39 ± 0.81 vs 12.85 ± 0.89 vs 8.31 ± 1.00, P < .01) as well as TNF-α (15.97 ± 0.81 vs 18.62 ± 2.35 vs 11.36 ± 0.82, P < .01) in vivo, whereas no obvious difference was found between the NT and sh-NC groups (Figure 5A-E). Furthermore, knockdown of NONO also significantly augmented the protein levels of IL-1β (1 vs 0.98 ± 0.05 vs 0.57 ± 0.09, P < .05), MCP-1 (1 vs 0.98 ± 0.05 vs 0.64 ± 0.11, P < .05) as well as TNF-α (1 vs 0.96 ± 0.10 vs 0.02 ± 0.01, P < .01) in VSMCs with Ang II stimulation (Figure 5F). These outcomes demonstrated that silencing of NONO participated in inhibiting the inflammation infiltration both in vivo and in vitro.

3.8 Effect of NONO on NF-κB translocation as well as phosphorylation in VSMCs with Ang II stimulation

Transcription factor NF-κB has an imperative part in the pathological course of atherosclerosis through regulating inflammatory cytokines, for instance IL-1β, IL-6 as well as MCP-1.10, 17 We implemented co-immunoprecipitation assays with anti-NONO or anti-NF-κB p65 antibodies in VSMCs following Ang II stimulation. We recognized that Ang II suggestively enhanced the interface of NONO plus NF-κB p65 (Figure 6A). We further established that knockdown of NONO significantly suppressed the nuclear translocation of NF-κB p65 in VSMCs with Ang II stimulation by double immunofluorescence histochemistry (Figure 6B). IgG was used as negative control. In addition, NONO silencing significantly suppressed the phosphorylation of NF-κB p65 in VSMCs stimulated with Ang II (1 vs 0.99 ± 0.03 vs 0.45 ± 0.07, P < .01, Figure 6C). Therefore, NONO might be co-immunoprecipitated with NF-κB and NONO knockdown suppressed the nuclear translocation of NF-κB p65, phosphorylation and activity.