Animal experiments were approved by the Institutional Animal Care and Use Committee of Hebei Medical University (approval ID: HebMU 20080026). Eight-week-old C57BL/6N male mice were purchased from Vital River Laboratory Animal Technology Co., Ltd., (Beijing, China). Animals were housed in a climatically controlled environment, on a 12 h light/dark cycle, with free access to water and standard food ad libitum.

The mice carotid artery ligation model applied has been described previously (21). Briefly, C57BL/6N male mice were anesthetized with 1.5% isoflurane. The left common carotid arteries were exposed and completely ligated with a 6–0 silk suture under the left carotid artery bifurcation to induce intima formation. The silk suture was passed below the exposed left carotid artery but not tightened as the control (n = 10). E2 (Sigma, 50-28-2, Purity ≥98%) (0.02 mg·kg⁻¹·day⁻¹) was infused through subcutaneous osmotic minipump (Alzet, Model 2004, USA) implantation 7 days before ligation injury and continuing for 14 days thereafter (n = 10). Ligated animals without E2 treatment received DMSO and corn oil at an equivalent amount (n = 10). The pcDNA3.1-BHLHE40 plasmids (n = 10) or pcDNA3.1-vehicle plasmids (n = 10) were diluted with Entranster™ solution (Engreen Biosystem, Beijing, China) and 10% glucose mixture (1:1 v/v) to 0.5 μg/μL in vivo. Then, added 10 μL aforesaid mixture into the 90 μL of 20% F-127 pluronic gel (Sigma, 9003-11-6) at 4°C for 2 h. Immediately after ligation, the exposed carotid artery adventitial surface was treated with 100 μL pluronic gel containing plasmids. At 14 days after surgery, all animals were anesthetized and perfused with cold PBS, and tissues were harvested for follow-up experiments.

For morphometric analyses, the arteries were fixed with 4% paraformaldehyde and embedded in paraffin. Four μm cross-sections were cut from the proximal carotid ligation site and prepared for hematoxylin and eosin (HE) staining. For each section, six random non-contiguous microscopic fields were analyzed. The neointimal area and intima-to-media ratio were calculated using Image-Pro Plus Analyzer (version 5.1) software (Media Cybernetics, Silver Spring, MD) in a blinded manner.

Mouse aortic vascular smooth muscle cell (mVSMC) (ATCC, No. CRL-2797™) were cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM, Gibco Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum (GEMINI, USA) and 1 × Penicillin-Streptomycin-Glutamine (Gbico, USA), containing 100 units/mL of penicillin and 100 μg/mL of streptomycin, cultured at 37°C with 5% CO₂ atmosphere. VSMCs were blocked by incubation in serum-deprived DMEM at 80–90% confluence or 24 h before stimulated with TNF-α or E2.

siRNAs targeting mouse BHLHE40 (si-BHLHE40) and negative control (si-Ctrl) were designed and synthesized by GenePharma (Shanghai, China). The siRNA sequences used in our studies were as follows:

The expression plasmids of BHLHE40 (pcDNA3.1-BHLHE40) were created by the placement of mouse BHLHE40 CDS region of mRNA into the pcDNA3.1 vector. The siRNAs or plasmids were transiently transfected into VSMC with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols.

The cell number was determined by Countess™ Automated Cell Counter (Invitrogen). After different treatment, VSMCs were digested, resuspended and blown into its individual tube. Ten μL of the cell suspension was mixed with 10 μL of Trypan blue, and counted by an Invitrogen Countess. Untreated cells were used for the baseline count. Each sample was counted three times, and the average value from triplicate experiments was measured.

Cell viability was determined using the MTS assay, as previously described (22). In brief, 1 × 10 ⁴ cells/well were seeded into 96-well plates with 5 replicates for each group, The next day, the cells were pretreated in 100 μL serum-free medium for 24 h and then incubated with appropriate treatment. The cells were incubated with CellTiter 96 AQueous One Solution (Promega, G3582) for 3 h, and the absorbance at 490 nm was measured using a Multiskan Spectrum (Thermo).

Total RNA was extracted from VSMC or mouse aortic tissues using Trizol (Invitrogen™) according to the manufacturer's instruction. The concentration and purity of the extracted RNA were detected by NanoDrop ND-2000 spectrophotometer (Thermo Fisher, Waltham, USA).cDNA was synthesized using an M-MLV First Strand Kit (Life Technologies) and real-time PCR analysis was done with the BIO-RAD CFX96^TM Real-Time System, using the Platinum SYBR Green qPCR SuperMix UDG Kit (Invitrogen). Relative mRNA expression levels were normalized to 18S. All PCRs were performed in triplicate. Relative amount of transcripts was calculated using the 2^−ΔΔCt formula.

The primer sequences were as follows:

Immunofluorescence staining was performed on 4 μm paraffin cross-sections from mouse artery samples. The sections were deparaffinized with xylene and rehydrated, and then were permeabilized by incubation with 0.5% Triton X-100 in phosphate-buffered saline (PBS). Non-specific sites were blocked by incubation in 10% normal goat serum (710027, KPL, USA) for 30 min. Then the sections were incubated with primary antibodies at 4°C overnight. The primary antibodies were mouse anti-SMα-actin (sc-130617, Santa Cruz) and rabbit anti-BHLHE40 (NB100-1800, Novus). Secondary antibodies were rhodamine-labeled antibody to rabbit IgG (031506, KPL, USA) and fluorescein-labeled antibody to mouse IgG (021815, KPL, USA). Nuclei were stained with DAPI (0100-20, SouthernBiotech) in each experiment. Images were captured by confocal microscopy (DM6000 CFS, Leica) and processed by LAS AF software.

Immunohistochemical staining was visualized by use of an SPN-9001 Histostain^TM-SP kit (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer's instruction. Paraffin cross-sections were deparaffinized with xylene and rehydrated in a graded ethanol series, and endogenous peroxidase activity was inhibited by incubation with 3% H₂O₂ for 30 min. Sections were blocked with 10% normal goat serum for 10 min and incubated overnight at 4°C with anti-BHLHE40 antibody (1:100 dilution, NOVUS, NB100-1800). After a PBS wash, sections were incubated with secondary antibody at 37°C for 30 min. Drops of horseradish enzyme labeled streptomycin were added for 15 min, washed with PBS for 5 min and three times and then DAB staining was performed under the ordinary light microscope. Sections were counterstained with hematoxylin to visualize nuclei.

The intracellular ROS levels were measured following the instruction of Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, China). Briefly, the cells were seeded in 12-well plates with microscope cover glasses and exposed to various treatments. The treated cells were loaded with 10 μM/L DCFH-DA at 37°C for 20 min. Subsequently, cells were washed with PBS three times and then observed using fluorescence microscopy (Olympus).

Protein was isolated from VSMC or aortic tissues as the manufacturer's instruction of RIPA Lysis Buffer (Solarbio, Beijing, China). Equal amounts of protein were electrophoresed on 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore). Membranes were blocked with 5% milk in TBS-Tween-20 (TBST) for 1.5 h at 37°C and incubated overnight at 4°C with the following primary antibodies: anti-PCNA (1:1000, ab92552, Abcam), anti-cyclin D1 (1:1000, 60186-1-Ig, Proteintech), anti-NOX1 (1:500, DF8684, Affinity Biotech), anti-NOX4 (1:500, 14347-1-AP, Proteintech), anti-p47^phox (1:1000, 4312, Cell Signaling Technology), anti-KLF4 (1:1000, GTX101509, GeneTex), anti-BHLHE40 (1:500, 17895-1-AP, Proteintech), anti-p44/42 MAPK (ERK1/2) (1:1000, 9102, Cell Signaling Technology), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000, 4370, Cell Signaling Technology), anti-JNK (1:500, 9252, Cell Signaling Technology), anti-phospho-SAPK/JNK (Thr183/Tyr185) (1:500, 4668, Cell Signaling Technology), anti-p38 MAPK (1:500, 9212, Cell Signaling Technology), anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500, 4511, Cell Signaling Technology) and anti-β-actin antibody (1:2000, ab6276, Abcam). Membranes were then incubated with secondary antibody (1:10000, Rockland) for 1.5 h at room temperature. At last, protein blots were treated with the Immobilon^TM western chemiluminescent HRP substrate (Millipore) and detected by ECL (enhanced chemiluminescence) Fusion Fx (Vilber Lourmat). Images were captured and processed by FusionCapt Advance Fx5 software (Vilber Lourmat).

The EdU incorporation assay was carried out according to the manufacturer's instruction (RiboBio, China). The representative images acquired by fluorescence microscope (Olympus). The cell proliferative rate was calculated as the proportion of Hoechst 33342-staining cells that incorporated EdU in 10 high-power fields per well.

Data are expressed as the means ± S.E.M. of at least three independent experiments. All analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA). Differences between two groups were analyzed by Student's t-test. For multiple comparisons or repeated measurements, ANOVA or repeated ANOVA followed by a Tukey's post-hoc test was used. A value of p < 0.05 was considered statistically significant.

HE staining showed that carotid arterial intima thickness was significantly increased in ligation injury-induced intimal hyperplasia mice models at 14 days post-operation. Compared with the ligated group, the degree of neointimal formation was obviously reduced in E2-treated group (Figure 1A). The ratio of intima to media (I/M ratio) and intimal area were dramatically lower in E2-treated group than that in the ligated group (Figures 1B,C). These results indicate that E2 can effectively inhibit neointimal formation induced by carotid artery ligation. Since it is known that ligation injury-induced intimal hyperplasia is closely related to VSMC proliferation and oxidative stress, we next investigate the effects of E2 on proliferation and oxidative stress-related genes expression in carotid arteries. Western blotting analysis revealed that vascular injury increased the expression of PCNA, cyclin D1, NOX1, NOX4 and p47^phox, whereas KLF4 expression was remarkably downregulated. Notably, carotid artery ligation-induced these changes were reversed by E2 (10 mg ·kg⁻¹·day⁻¹) treatment (Figure 1D). qRT-PCR analysis of PCNA, cyclin D1, NOX1, NOX4, p47^phox and KLF4 expression was consistent with their expression of protein level (Figure 1E). Overall, these studies demonstrated E2 could alleviate vascular remodeling in intimal hyperplasia mice partly through limiting the proliferation and oxidative stress of VSMC.

Because it is known that TNF-α stimulates VSMC proliferation and oxidative stress, we sought to determine whether E2 suppressed neointimal hyperplasia through restraining TNF-α-induced VSMC proliferation and oxidative stress. As shown in Figures 2A–D, TNF-α treatment markedly increased VSMC viability and number in a dose and time-dependent manner by MTS assay and cell counting. Simultaneously, exposure of VSMC to TNF-α dose and time-dependently enhanced mRNA and protein expression of PCNA, cyclin D1, NOX1, NOX4 and p47^phox (Figures 2E–H). Next, we detected the effects of E2 treatment on VSMC proliferation and oxidative stress induced by TNF-α. As shown by MTS assay and cell counting, treating VSMC with TNF-α (10 ng/mL) promoted cell proliferation in a time-dependent manner, whereas pretreatment of VSMC with 25, 50 and 100 nM of E2 for 6 h dose-dependently abrogated the inducing effects of TNF-α on VSMC viability and number (Figures 2I,J). Western blotting and qRT-PCR assay displayed that E2 offsets the up-regulation of PCNA, cyclinD1, NOX1, NOX4 and p47^phox expression induced by TNF-α (Figures 2K,L). In addition, EdU staining proved that E2 reversed TNF-α-induced VSMC proliferation (Figures 2M,N). In Figure 2O, E2 also visibly blocked TNF-α-induced ROS production in VSMC. In general, these results indicate that E2 inhibits TNF-α-induced VSMC proliferation and oxidative stress.

In order to obtain which genes have been changed during neointimal hyperplasia, we downloaded an expression dataset (GSE56143) from the Gene Expression Omnibus (GEO), and found that the rhythm gene BHLHE40 was down-regulated in the ligated vascular tissue (Figure 3A). It has been reported that BHLHE40 can participate in the occurrence and development of cancer (23), but its role in the regulation of proliferation and oxidative stress in VSMC is still unclear. Therefore, we focused our research on BHLHE40. Western blotting and qRT-PCR assay showed that compared with unligated tissues, protein and mRNA expression levels of BHLHE40 were down-regulated by more than 0.5 times at 14 days after carotid artery ligation (Figures 3B,C). Furthermore, both immunofluorescence staining and immunochemistry staining of BHLHE40 were markedly reduced in injured arteries compared to sham-operation. Noticeably, carotid artery ligation-induced downregulation of BHLHE40 was reversed by E2 (Figures 3D,E). Western blotting (Figure 3F) and qRT-PCR assay (Figure 3G) revealed that TNF-α treatment lessened protein and mRNA expression of BHLHE40 compared with the control group, whereas pretreatment with E2 (100 nM) largely counteracted the inhibitory effects of TNF-α on BHLHE40 expression. Taken together, these findings suggest that E2 promotes the expression of BHLHE40 both in vivo and in vitro.

To further illustrate the role of BHLHE40 in ligation injury-induced intimal hyperplasia, we assayed the effects of BHLHE40 down-regulation on cellular proliferation and oxidative stress in VSMC. Firstly, we confirmed that the expression of BHLHE40 at the protein and mRNA levels was silenced by about 70% in si-BHLHE40 transfected VSMC (Figure 4A). Subsequently, we examined the effects of si-BHLHE40 on the expression of proliferation and oxidative stress-related genes, and found that treating VSMC with TNF-α clearly increased the expression of PCNA, cyclinD1, NOX1, NOX4 and p47^phox, which was enforced by si-BHLHE40 transfection (Figures 4B,C). In follow-up experiments, we found that BHLHE40 knockdown increased TNF-α-induced proliferation in VSMC, as shown by MTS analysis and cell counting (Figures 4D,E). Meanwhile, EdU staining evidenced that depletion of BHLHE40 by its siRNA increased TNF-α-induced VSMC proliferation (Figures 4F,G). In Figure 4H, ROS staining showed that si-BHLHE40 and TNF-α co-treatment further enhanced TNF-α-induced ROS production in VSMC. All in all, these data suggested that knockdown of BHLHE40 contributes to TNF-α-induced VSMC proliferation and oxidative stress.

Next, we successfully overexpressed the BHLHE40 at both mRNA and protein level in VSMC (Figure 5A). To further explore whether BHLHE40 participates in the induction of proliferation and oxidative stress in TNF-α-treated VSMC, we forcedly expressed BHLHE40 and found that BHLHE40 overexpression distinctly reduced the expression of PCNA, cyclinD1, NOX1, NOX4 and p47^phox induced by TNF-α at both mRNA and protein levels (Figures 5B,C). As presented by MTS assay and cell counting, overexpression of BHLHE40 efficaciously counteracted the stimulatory effect of TNF-α on VSMC proliferation (Figures 5D,E). Similarly, EdU staining showed that the enforced expression of BHLHE40 in VSMC had opposite effects on TNF-α-induced proliferation (Figures 5F,G). Up-regulation of BHLHE40 led to a decrease in the production of TNF-α-induced ROS (Figure 5H). Altogether, these results indicate that BHLHE40 negatively regulates the proliferation and oxidative stress of VSMC by affecting the expression of PCNA, cyclinD1, NOX1, NOX4 and p47^phox.

Next, we performed BHLHE40 knockdown experiment to investigate whether BHLHE40 mediates the inhibitory role of E2 in the proliferation and oxidative stress of VSMCs. As shown in Figure 6A, down-regulation of BHLHE40 can reverse the inhibitory effects of E2 on the proliferation and oxidative stress. It is known that MAPK cascade activation is the center of multiple signaling pathways, and plays a key role in cell proliferation, inflammation and oxidative stress. Western blotting analysis revealed that TNF-α treatment markedly increased phosphorylation of ERK, JNK and P38 in VSMC, but the effects of TNF-α on MAPK signaling pathways were normalized by E2 treatment (Figure 6B). In order to clarify the mechanism by which BHLHE40 regulates proliferation and oxidative stress, we up-regulated or down-regulated the expression of BHLHE40 in VSMC, and monitored the expression of related genes in the MAPK signaling pathway. As shown in Figure 6C, up-regulation of BHLHE40 can lead to decreased ERK, JNK and p38 phosphorylation. On the contrary, down-regulating the expression of BHLHE40 can usefully increase ERK, JNK and p38 phosphorylation (Figure 6D). In order to confirm whether E2 regulates the MAPK signaling pathway by affecting the expression of BHLHE40, we conducted rescue experiments. As demonstrated in Figure 6E, TNF-α-induced phosphorylation of ERK, JNK and P38 were partly inhibited after E2 preincubation (Figure 6E, lane 3 vs. lane 2). Knockdown of BHLHE40 restrained this inhibitory effect of E2 (Figure 6E, lane 4 vs. lane 3). In addition, we examined the effect of E2 and BHLHE40 on AKT phosphorylation, as shown in Supplementary Figure S1, E2 treatment can lead to decreased AKT phosphorylation, but down-regulated BHLHE40 have no influence on the inhibitory effect of E2. On balance, the above results confirmed that 100 μM E2 displays suppressive effects on TNF-α-induced pathologic changes through deactivating MAPK signal pathways.

To examine whether BHLHE40 is a key mediator in vascular remodeling, Pluronic F-127 gel solution containing pcDNA3.1 plasmids or pcDNA3.1-BHLHE40 plasmids were applied to the exposed adventitial surface of an ~5 mm segment of the ligated carotid artery. The intimal thickness of the ligated artery was determined 14 days after the surgery. As expected, carotid arterial ligation increased vascular wall thickness in control-plasmids transfected mice, and this expansion was strongly reduced in BHLHE40-plasmids transfected mice (Figure 7A). Consistent with these results, BHLHE40-overexpressed mice showed an important decrease in the ratio of intimal/medial area (I/M ratio) and neointimal area compared with control-plasmids transfected mice (Figures 7B,C). Next, we examined the expression of PCNA, cyclinD1, NOX1, NOX4 and p47^phox and KLF4 in the injured carotid artery of pcDNA3.1 plasmids or pcDNA3.1-BHLHE40 plasmids transfected mice. Notably, western blotting and qRT-PCR analysis data showed that carotid artery ligation-induced above gene changes were normalized by BHLHE40 overexpression (Figures 7D,E). To sum up, these data support the pathophysiological role of BHLHE40 depletion in vascular hypertrophy.