Nuclear receptor subfamily 1 group D member 1 suppresses the proliferation, migration of adventitial fibroblasts, and vascular intimal hyperplasia via mammalian target of rapamycin complex 1/β-catenin pathway

ABSTRACT

Background

In-stent restenosis hardly limits the therapeutic effect of the percutaneous vascular intervention. Although the restenosis is significantly ameliorated after the application of new drug-eluting stents, the incidence of restenosis remains at a high level.

Objective

Vascular adventitial fibroblasts (AFs) play an important role in intimal hyperplasia and subsequent restenosis. The current study was aimed to investigate the role of nuclear receptor subfamily 1, group D, member 1 (NR1D1) in the vascular intimal hyperplasia.

Methods and Results

We observed increased expression of NR1D1 after the transduction of adenovirus carrying Nr1d1 gene (Ad-Nr1d1) in AFs. Ad-Nr1d1 transduction significantly reduced the numbers of total AFs, Ki-67-positive AFs, and the migration rate of AFs. NR1D1 overexpression decreased the expression level of β-catenin and attenuated the phosphorylation of the effectors of mammalian target of rapamycin complex 1 (mTORC1), including mammalian target of rapamycin (mTOR) and 4E binding protein 1 (4EBP1). Restoration of β-catenin by SKL2001 abolished the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs. Surprisingly, the restoration of mTORC1 activity by insulin could also reverse the decreased expression of β-catenin, attenuated proliferation, and migration in AFs induced by NR1D1 overexpression. In vivo, we found that SR9009 (an agonist of NR1D1) ameliorated the intimal hyperplasia at days 28 after injury of carotid artery. We further observed that SR9009 attenuated the increased Ki-67-positive AFs, an essential part of vascular restenosis at days 7 after injury to the carotid artery.

Conclusion

These data suggest that NR1D1 inhibits intimal hyperplasia by suppressing the proliferation and migration of AFs in a mTORC1/β-catenin-dependent manner.

KEYWORDS:

• NR1D1
• mTORC1
• β-catenin
• AFs
• neointimal hyperplasia

Introduction

Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality worldwide (1,2). Percutaneous vascular interventions are a well-established therapeutic strategy for the treatment of patients with coronary, carotid, or peripheral vascular atherosclerotic diseases (3,4). Vascular restenosis, a major drawback of intravascular interventions, leads to adverse cardiovascular events (5). Restenosis results from multiple factors such as vascular constrictive remodeling and intimal hyperplasia. Vascular fibroblasts from the adventitia and vascular smooth muscle cells (VSMCs) from the media migrate into the intima and proliferate, finally causing intimal hyperplasia (6). Recent studies have shown adventitial fibroblasts (AFs) play an important role in vascular remodeling (7,8) and intimal hyperplasia (6). However, the underlying mechanism of how AFs are involved in intimal hyperplasia is unclear. Therefore, revealing the potential mechanism of AFs in vascular restenosis is of great importance for finding new therapeutic targets.

Circadian system, driven by molecular clocks, plays important roles in the regulation of cardiovascular physiology and diseases (9). Widely expressed in different cell types, molecular clocks regulate various pathological and physiological functions (10). As a member of the nuclear receptor family, nuclear receptor subfamily 1, group D, member 1 (NR1D1) is a transcription inhibitor and a core component of molecular clocks (11). NR1D1 and retinoid-related orphan receptor α represses and activates brain and muscle ARNT-like 1 (Bmal1) transcription, respectively, via competing for the binding site of ROR response element in the Bmal1 promoter (12). NR1D1 is involved in numerous physiological and pathophysiological functions, including lipid metabolism, autophagy, mitochondrial biogenesis, and inflammation (13,14). NR1D1 plays a protective role in the development of atherosclerosis, heart failure, and myocardial infarction (15–17). Previous studies have shown that NR1D1 inhibits the proliferation of breast cancer cells (18), ovarian cancer cells (19), and embryonic fibroblasts (12). However, the role of NR1D1 in the proliferation, migration of AFs, and intimal hyperplasia remains unknown.

Wnt/β-catenin signaling plays a prominent role in cellular differentiation, proliferation, and migration (20). Activation of Wnt signaling pathway promotes the release and accumulation of β-catenin. When β-catenin rises to a certain concentration, it transfers into the nucleus to activate the expression of genes involved in cell cycle progression (13,21). In addition, β-catenin is involved in the proliferation of vascular endothelial cells, smooth muscle cells, and intimal hyperplasia (22,23). Recently, NR1D1 is confirmed to inhibit the proliferation of 3T3-L1 preadipocytes and bone mesenchymal stem cells via suppressing β-catenin (13,24). However, whether β-catenin is involved in NR1D1-mediated regulation of AFs is unrevealed.

In the current study, we investigated the role of NR1D1 in the proliferation, migration of AFs, and vascular intimal hyperplasia. In vitro, NR1D1 overexpression inhibited the proliferation and migration of AFs via decreasing the expression of β-catenin. Mammalian target of rapamycin complex 1 (mTORC1) participated in NR1D1-mediated downregulation of β-catenin in AFs. Additionally, activation of NR1D1 ameliorated intimal hyperplasia after carotid artery injury via inhibiting the early-stage proliferation and migration of AFs in vivo.

Materials and methods

Experimental animals

The animal experiments in this experiment were approved by Institutional Animal Care and Use Committee and the Ethic Committee of The General Hospital of Western Theater Command (Chengdu, Sichuan, China). Male C57BL/6 J mice at the age of 8–10 weeks old were purchased from Hunan SJA Laboratory Animal Co., Ltd (Changsha, Hunan, China). The housing conditions were maintained at room temperature, free access to water and food, and with a 12 h dark/light cycle. The left common carotid artery of the mouse was injured by the insertion of a 0.14-mm guide wire as described in the previous literature (4). SR9009 (100 mg/kg; MCE, Monmouth Junction, NJ, USA) was administered twice a day via intraperitoneal injection for consecutive 7 days immediately after injury (25). The mice were euthanized by anesthesia (pentobarbital, 100 mg/kg) 7 or 28 days after the operation. The carotid artery was isolated for further experiments.

Culture and treatment of mouse adventitial fibroblasts

Culture of AFs was prepared as described in the literature (26). Briefly, the thoracic aorta was isolated from C57BL/6 J mice after anesthesia. The outer membrane tissue was separated and cut into pieces. These tissue fragments were then incubated at 37°C in 5% CO₂ atmosphere and cultured in complete medium [Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Carlsbad, CA, USA) supplemented with fetal calf serum (10%; Gibco, Carlsbad, CA, USA), penicillin (100 units/mL), and streptomycin (100 mg/mL)] for 4 days. The culture medium was replaced every other day. About 7 days later, the primary cells reached 80-90% confluence. The cells in passage 4–8 were used in the further experiments. SKL2001 (40 uM, MCE) and insulin (5 mg/L, Solarbio, Beijing, China) were utilized to treat the cultured AFs.

Adenoviral transduction

AFs in passage 4 were cultured in 6-well plates with an initial density of 1 × 10⁵/mL. 24 h later, AFs were transfected with the adenovirus carrying Nr1d1 gene (Ad-Nr1d1) or control empty vector (Ad-Con) with a multiplicity of infection (MOI) of 50. 8–12 h after transfection, the transfection medium was replaced with complete medium.

Immunofluorescence

For immunofluorescence staining of AFs, the 24-well plates filled with AFs were washed with phosphate buffered solution (PBS) three times after removing the cell medium. Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized in ddH₂O containing 0.5% Triton X-100 for 10 min, and then blocked with 10% bovine serum albumin for 30 min at room temperature. Subsequently, cells were incubated with Ki-67 primary antibody (1:200, Bioss, Beijing, China) overnight at 4°C. Next day, cells were incubated with CoraLite488-conjugated goat anti-rabbit secondary antibody (1:500, Proteintech, Beijing, China) for 1 h at dark after washing. Washed with PBS for three times, the cells were counterstained with DAPI (Solarbio) for 3 min. The images were captured by immunofluorescent microscopy (Leica MPS 60; Wetzlar, HD, Germany). For immunofluorescence of carotid artery sections, the tissue slices were incubated with antibodies against to Ki-67 (1:200, Bioss), vimentin (1:200, Proteintech), or periostin (1:4000, Proteintech) overnight at 4°C. Next day, cells were incubated with CoraLite 488-conjugated goat anti-rabbit secondary antibody (1:500, Proteintech) and Cy3–conjugated affinipure goat anti-mouse (1:100, Proteintech) secondary antibody for 1 h in dark. Then, the slices were counterstained with DAPI after washing. The images were captured by immunofluorescent microscopy (Leica MPS 60).

Immunohistochemical staining

Standard hematoxylin and eosin (HE) and Ki-67 staining was used to analyze histological changes of carotid arteries. Slices were randomly selected from all four groups. For HE staining, the 4-μm slices were stained with hematoxylin and eosin after dewaxing and hydration. Finally, Image-Pro Plus software was used to analyze the ratio of intima to media. For Ki-67 staining, slices were incubated with the primary antibody against Ki-67 (Bioss) overnight at 4°C after being blocked by 10% BSA. Next day, the slices were incubated with corresponding secondary antibody and counterstained with hematoxylin. Finally, the slices were observed under a microscope (Olympus VS200, Tokyo, Japan).

Western blotting

After lysing, centrifugation, and denaturalization, the samples were added to the lanes for electrophoresis. Then, the samples were separated and transferred to polyvinylidene fluoride membrane (Millipore, Burlington, MA) membranes. After blocked with 5% BSA for 1 h, the membranes were incubated with primary antibodies against NR1D1, β-catenin, 4EBP1, p-4EBP1, mTOR, p-mTOR, GAPDH (1:1000 Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. After washing, membranes were incubated with corresponding secondary antibodies (1:10,000) at room temperature for 1 h. Finally, membranes were incubated with enhanced chemiluminescence and subjected to exposure. The band intensities were analyzed by Image J software (NIH, Bethesda, MD).

Cell counting kit (CCK)-8 assay

Cell counting kit (Solarbio) was utilized as described by the manufacturer’s instruction. Briefly, AFs were cultured in 96-well plastic plates in complete media at an initial density of 5 × 10⁴/mL. After treatment of SKL2001 or insulin, 10 uL of CCK-8 reagent was added to each well and incubated with AFs at 37°C for another 2 h. Finally, the viability of AFs was determined by Enzyme Labeling Instrument (Thermo Fisher Scientific, Waltham, MA, USA) via calculating the relative absorbance at 450 nm.

Wound-healing assay

AFs were planted in 6-well plates (1 × 10⁵/mL cells per well) and serum-deprived for 24 h. A scratch was drawn with a 200 μL sterile pipette in the middle area of the cells. Washed with PBS for three times, the images of cells were captured at corresponding time points.

Statistical analysis

The data were analyzed using GraphPad Prism 9 and presented as mean ± SD. The difference between the two independent groups was evaluated by unpaired Student’ t-test. The difference among three or more groups was assessed by a one-way analysis of variance with an appropriate post hoc test. P< .05 was considered statistically significant.

Results

NR1D1 suppresses the proliferation and migration of mouse AFs

To explore the underlying role of NR1D1 in the proliferation and migration of AFs, we utilized Ad-Nr1d1 transduction to overexpress NR1D1 in AFs. We found that the expression of NR1D1 was significantly increased after Ad-Nr1d1 transduction (Figure 1a,b). CCK-8 assay showed that the cell viability was decreased after Ad-Nr1d1 transduction, suggesting that NR1D1 might inhibit the proliferation or enhance the apoptosis of AFs (Figure 1c). Furthermore, NR1D1 overexpression significantly reduced the percentage of Ki-67-positive AFs (Figure 1d,e), indicating that the proliferation of AFs was attenuated after NR1D1 overexpression. In addition, we also examined the role of NR1D1 in the migration of AFs. Wound-healing assay showed that NR1D1 overexpression delayed the healing rate of AFs (Figure 1f,g). These data indicate that NR1D1 inhibits the proliferation and migration of mouse AFs.

Figure 1. NR1D1 overexpression inhibits the proliferation and migration of AFs. (a) Representative western blotting bands of NR1D1 in AFs transfected with Ad-Con or Ad-Nr1d1. (b) Quantification of western blotting bands for NR1D1 in AFs (n = 4). (c) The viability of AFs transfected with Ad-Con or Ad-Nr1d1 was assessed by CCK-8 assay. The absorbance was obtained at 450 nm (n = 5). (d) Representative pictures of AFs stained with Ki-67 (green) and DAPI (blue) after transfection with Ad-Con or Ad-Nr1d1. (e) Quantification of Ki-67-positive AFs (n = 4). Magnification 400 × . (f) Migration of AFs transfected with Ad-Con or Ad-Nr1d1 was measured by wound healing assay. (g) Quantification of wound-healing rates in AFs (n = 5). Magnification 100 × . Data are shown as mean ± S.D. ***P < .001 versus Ad-Con.

NR1D1 inhibits the proliferation and migration of mouse AFs via decreasing the expression of β-catenin

β-Catenin plays a vital role in regulating vascular functions. β-Catenin regulates the proliferation of multiple cell types such as vascular endothelial cells and VSMCs (22). To investigate whether β-catenin was involved in NR1D1-mediated regulation of proliferation and migration in AFs, we first examined the effect of NR1D1 on the expression of β-catenin. As shown in our data, NR1D1 overexpression reduced the protein level of β-catenin, which was reversed by SKL2001 (Figure 2a,b). We further confirmed whether the restoration of β-catenin by SKL2001 could abolish the inhibitory effects of NR1D1 on the proliferation and migration of mouse AFs. We found that the inhibitory effects of NR1D1 on the cell viability (Figure 2c), percentage of Ki-67-positive AFs (Figure 2d,e) and wound-healing rate of AFs (Figure 2f,g) were both attenuated by the application of SKL2001. These results demonstrate that NR1D1 suppresses the proliferation and migration of mouse AFs via inhibiting the expression of β-catenin.

Figure 2. The inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs are attenuated by SKL2001. AFs were transfected with Ad-Con or Ad-Nr1d1 and then incubated with or without SKL2001. (a) Representative western blotting bands of β-catenin and NR1D1 in AFs treated as above mentioned. (b) Quantification of western blotting bands for β-catenin in AFs (n = 4). (c) The viability of AFs treated as above mentioned was assessed by CCK-8 assay. The absorbance was obtained at 450 nm (n = 5). (d) Representative pictures of AFs stained with Ki-67 (green) and DAPI (blue) as above treated. (e) Quantification of Ki-67-positive AFs (n = 4). Magnification 400 × . (f) Migration of AF treated as above mentioned were measured by wound healing assay. (g) Quantification of wound healing rates in AFs (n = 5). Magnification 100 × . Data are shown as mean ± S.D. LSD test for B, C, E and G. ***P < .001 versus Ad-Con; ^###P < .001 versus Ad-Nr1d1.

NR1D1-mediated inhibition of β-catenin in AFs is reversed by re-activating mTORC1

mTORC1 is of great importance in regulating proliferation, migration of AFs, and neointimal hyperplasia (27). The translation of β-catenin is proved to be increased in a mTORC1-dependent manner (28). To investigate whether mTORC1 plays a role in NR1D1-mediated regulation of β-catenin in AFs, we first examined whether NR1D1 affected mTORC1 activity. The phosphorylation of mTOR and 4EBP1, the main effector of mTORC1, can reflect mTORC1 activity (29). As our data showed, NR1D1 suppressed the phosphorylation level of 4EBP1^Thr37/46 and mTOR^Ser2448 (Figure 3a,b). We next examined whether restoration of mTORC1 activity by insulin could abolish the inhibitory effect of NR1D1 on the expression of β-catenin. Interestingly, the inhibitory effect of NR1D1 on the expression of β-catenin was also reversed by insulin (Figure 3c,d), indicating that NR1D1 suppresses β-catenin by inhibiting mTORC1 activity.

Figure 3. NR1D1-mediated repression on β-catenin is abolished by activating mTORC1. (a) Representative western blotting bands of mTOR, p-mTOR, 4EBP1 and p-4EBP1 in AFs transfected with Ad-Con or Ad-Nr1d1. (b) Quantification of western blotting bands for p-mTOR/mTOR and p-4EBP1/4EBP1 in AFs (n = 5). AFs were transfected with Ad-Con or Ad-Nr1d1 and then incubated with or without insulin. (c) Representative western blotting bands of β-catenin and NR1D1 in AFs as above treated. (d) Quantification of western blotting bands for β-catenin in AFs (n = 4). Data are shown as mean ± S.D. LSD test for D. **P < .01 and ***P < .001 versus Ad-Con; ^##P < .01 versus Ad-Nr1d1.

We further wondered if the inhibitory effects of NR1D1 on the proliferation and migration of mouse AFs could be reversed by insulin. As our data showed, utilization of insulin attenuated the inhibition of NR1D1 on the cell viability (Figure 4a) and the percentage of Ki-67-positive AFs (Figure 4b,c). Simultaneously, the reduced wound-healing rate induced by NR1D1 overexpression was also elevated after re-activation of mTORC1 by insulin (Figure 4d,e). Our data demonstrate that NR1D1 suppresses the proliferation and migration of mouse AFs via mTORC1/β-catenin axis.

Figure 4. The inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs are abolished by activating mTORC1. AFs were transfected with Ad-Con or Ad-Nr1d1 and then incubated with or without insulin. (a) The viability of AFs as above treated was assessed by CCK-8 assay. The absorbance was obtained at 450 nm (n = 5). (b) Representative pictures of AFs stained with Ki-67 (green) and DAPI (blue) as above mentioned. (c) Quantification of Ki-67-positive AFs (n = 4). Magnification 400 × . (d) Migration of AFs treated as above mentioned was measured by wound healing assay. (e) Quantification of wound-healing rates in AFs (n = 5). Magnification 100 × . Data are shown as mean ± S.D. LSD test for A, C and E. ***P < .001 versus Ad-Con; ^###P < .001 versus Ad-Nr1d1.

NR1D1 ameliorates intimal hyperplasia after vascular injury

To investigate the crosstalk of NR1D1 and vascular intimal hyperplasia, we injured the left common carotid artery in the mouse to induce intimal hyperplasia and performed HE and Ki-67 immunohistochemical staining to explore the role of NR1D1 in intimal hyperplasia. HE staining showed that the vascular lumen was obviously narrower and the intima-to-media ratio was increased in arteries harvested at 28 days after injury, which was ameliorated by SR9009 (Figure 5a,b). Ki-67 staining showed that the enlarged Ki-67-positive area within intima of the injured carotid artery was also attenuated by SR9009 (Figure 5c,d). Our data demonstrate that NR1D1 ameliorates intimal hyperplasia after vascular injury.

Figure 5. NR1D1 ameliorates intimal hyperplasia after vascular endothelial injury in mice. (a) Representative HE staining of carotid arteries from C57BL/6 J mice with or without SR9009 (100 mg/kg) treatment at day 28 after sham operation or wire injury. (b) Quantification for ratio of intima/media (n = 5). Magnification 200 × . (c) Representative immunohistochemical staining of Ki-67 (brown) in sections of carotid arteries from C57BL/6 J mice with or without SR9009 treatment at day 28 after sham operation or wire injury. (d) Quantification for Ki-67-positive cells within neointima (n = 5). Magnification 200 × . Data are shown as mean ± S.D. LSD test for B and D.***P < .001 versus Sham+DMSO; ^###P < .001 versus Injured+DMSO.

NR1D1 suppresses the proliferation of AFs at an early stage after injury

A previous study provides direct evidence that early-stage AFs after injury migrate from adventitia to intima and proliferate, thus becoming a new source of subsequent neointima (8). To explore the crosstalk of NR1D1-mediated regulation on AFs and intimal hyperplasia, we utilized immunofluorescence staining of Ki-67 and α-SMA (a marker of VSMC) in arteries 7 d after injury to determine the cell proliferation within adventitia. Immunofluorescence of Ki-67 and α-SMA revealed that the adventitial layer was thickened and the number of Ki-67-positive cells within adventitia was significantly increased 7 d after injury. The application of SR9009 significantly attenuated the adventitial thickening and decreased the increased number of Ki-67-positive cells within adventitia induced by injury (Figure 6a,b). Next, we performed co-staining of Ki-67 with vimentin or periostin as markers for AFs. The results showed that the ratio of both Ki-67⁺/vimentin⁺ and Ki-67⁺/periostin⁺ AFs within adventitia were increased 7 d after injury, which were attenuated by SR9009 (Figure 6c,d). Our data indicate that NR1D1 ameliorates intimal hyperplasia partly by attenuating the proliferation of AFs at an early stage after injury.

Figure 6. The early-stage proliferation of AFs within adventitia after injury is suppressed by NR1D1 in mice. (a) Representative immunofluorescence co-staining of Ki-67 (green) with α-smooth muscle actin (α-SMA, red) and DAPI (blue) from carotid arteries of C57BL/6 J mice with or without SR9009 treatment at day 7 after sham operation or wire injury. (b) Quantification for Ki-67-positive cells in adventitia (n = 4). Magnification 200 × . (c) Representative immunofluorescence co-staining of Ki-67 (green) with periostin (red) or vimentin (red) from carotid arteries of C57BL/6 J mice with or without SR9009 treatment at day 7 after sham operation or wire injury. (d) Quantification for the ratio of Ki-67-positive AFs (n = 4). Magnification 600 × . Data are shown as mean ± S.D. LSD test for B and D. ***P < .001 versus Sham+DMSO; ^###P < .001 versus Injured+DMSO.

Discussion

Intimal hyperplasia is a crucial process for ISR (5). Adventitia, an important part of the vascular wall, serves as an active participant in the vascular diseases (30). Recent research showed that AFs, the main component of vascular adventitia, were involved in intimal hyperplasia and vascular remodeling (30,31). Nevertheless, the underlying mechanisms are largely unknown. Our current study demonstrates that NR1D1 suppresses the proliferation and migration of AFs. NR1D1-mediated regulation of AFs depends on the downregulation of β-catenin and mTORC1. Inhibition of β-catenin by NR1D1 relies on the decreased activity of mTORC1. Importantly, NR1D1 also suppresses the early-stage proliferation of AFs within adventitia and subsequent intimal hyperplasia. Therefore, our data highlight a crucial role of NR1D1 in the treatment of intimal hyperplasia-associated vascular diseases.

As a core member of clock genes, NR1D1 is highly expressed in mammalian liver, skeletal muscle, brain, and adipose tissue and involved in cell differentiation, lipid metabolism, and inflammatory process (13). NR1D1 participates in the regulation of cardiovascular diseases. For example, SR9009 protects against acute myocardial ischemia via reducing cytokine production and inhibiting inflammatory cells infiltration (17). NR1D1 is also involved in modulating vascular diseases, including vascular repair (32), suppressing atherosclerosis (33), and promoting abdominal aortic aneurysm (34). Vascular intimal hyperplasia shares similar pathological process to atherosclerosis, such as abnormal migration and proliferation of VSMCs and AFs. Interestingly, we currently demonstrate that NR1D1 also ameliorates intimal hyperplasia after injury. However, the underlying molecular mechanism remains unclear.

Serving as the major component of vascular adventitia, AFs play a vital role in vascular function and structure (35). Recent studies have demonstrated that excessive migration and proliferation of AFs are also a critical source of intimal hyperplasia (36). Previous studies have demonstrated that NR1D1 inhibits proliferation in different cell types, including cancer cells, 3T3-L1 preadipocytes, and bone mesenchymal stem cells (13,24,37). However, the role of NR1D1 in the proliferation and migration of AFs is unclear. In the current study, we found that NR1D1 overexpression significantly suppressed the proliferation and migration of AFs. Thus, NR1D1-mediated regulation of AFs might be a potential target for vascular intimal hyperplasia.

Wnt is a classical signaling pathway that promotes the proliferation and migration of AFs (38). As the canonical effect factor of Wnt signaling, β-catenin is also associated with accelerated proliferation, migration of AFs (26), and intimal hyperplasia (39). NR1D1 suppresses the expression of β-catenin in 3T3-L1 preadipocytes and bone mesenchymal stem cells (13,24). Consistent with the above conclusions, we also observed a decreased expression of β-catenin in AFs after transduction of Ad-Nr1d1. Recovery of the expression of β-catenin by SKL2001 abolished the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs. Our data indicate that NR1D1 suppresses the proliferation and migration of AFs via reducing β-catenin expression.

mTOR is a serine/threonine protein kinase and exerts various roles by forming two complexes, mTORC1 and mTORC2 (40). mTORC1 is essential for cardiovascular events, including embryonic cardiovascular development, the maintenance of adult cardiac structure and function, and promoting the phenotypical transition in VSMCs and AFs (29,41). mTORC1 is reported to cause the accumulation of β-catenin in tumorigenesis (28). Whether NR1D1 regulates β-catenin expression in AFs in a mTORC1-dependent manner is unclear. In the current study, we found that the mTORC1 activity was notably reduced by NR1D1. Furthermore, the re-activation of mTORC1 by insulin abolished the decreased expression of β-catenin, attenuated proliferation, and migration of AFs induced by NR1D1. Our data demonstrate that NR1D1 suppresses the proliferation and migration of AFs via mTORC1/β-catenin pathway.

We have confirmed that NR1D1 inhibits the proliferation and migration of AFs in vitro. However, the regulation of NR1D1 on AFs within adventitia in vivo and its crosstalk with intimal hyperplasia is unrevealed. Recent studies have highlighted the important impact of adventitia on neointima formation. Early activation and proliferation of AFs within adventitia causes a robust expansion of the adventitial layer. These activated AFs secrets cytokines and inflammatory factors, thus stimulating subsequent phenotypical switching of VSMCs and intimal hyperplasia (31). In our current study, we confirmed that the thickened adventitial layer and increased proliferating AFs within adventitia at an early stage (7 d) after injury were both attenuated by NR1D1, which might partly illustrate how NR1D1-mediated regulation of AFs in vivo contributes to relief of the subsequent vascular intimal hyperplasia.

Conclusions

Taken together, we demonstrate that NR1D1 ameliorates intimal hyperplasia after injury partly by inhibiting the proliferation and migration of mouse AFs. These effects rely on the mTORC1/β-catenin pathway. Our current findings provide evidence that NR1D1 is a potential therapeutic target for treating vascular restenosis-associated diseases.

Author contributions

K.P. and M.L.W. contributed equally to the work. K.P. and M.L.W. conceived the project. D.C.Y. and X.S.S. designed the study. K.P., M.L.W., D.C.Y., X.S.S., and Y.J.Y. supervised the entire research. K.P. and M.L.W. performed most of the experimental work and conducted data analysis. J.W., Q.W., and D.L. provided some technical support. K.P. contributed to the figure preparation. D.C.Y. X.S.S., and Y.J.Y. discussed the study. D.C.Y. and X.S.S. organized the data and wrote the manuscript. All authors reviewed the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10641963.2023.2178659

Additional information

Funding

This work was supported by the Project of The General Hospital of Western Theater Command (2021-XZYG-A02 to D. Yang, 2021-XZYG-B27 to K. Peng, 2021-XZYG-A01 to Y. Yang, and 2021-XZYG-C41 to X. Sun), the National Natural Science Foundation of China (82100419 to X. Sun and 82070289 to Y. Yang) and Sichuan Science and Technology Program (2022NSFSC0820 to Q. Wang; Incubation Project of The General Hospital of Western Theater Command.