NR1D1 Deletion Induces Rupture-Prone Vulnerable Plaques by Regulating Macrophage Pyroptosis via the NF-κB/NLRP3 Inflammasome Pathway

Vulnerable plaque rupture is the main trigger of most acute cardiovascular events. But the underlying mechanisms responsible for the transition from stable to vulnerable plaque remain largely unknown. Nuclear receptor subfamily 1 group D member 1 (NR1D1), also known as REV-ERB α, is a nuclear receptor that has shown the protective role in cardiovascular system. However, the effect of NR1D1 on vulnerable plaque rupture and its underlying mechanisms are still unclear. By generating the rupture-prone vulnerable plaque model in hypercholesterolemic ApoE−/− mice and NR1D1−/−ApoE−/− mice, we demonstrated that NR1D1 deficiency significantly augmented plaque vulnerability/rupture, with higher incidence of intraplaque hemorrhage (78.26% vs. 47.82%, P = 0.0325) and spontaneous plaque rupture with intraluminal thrombus formation (65.21% vs. 39.13%, P = 0.1392). In vivo experiments indicated that NR1D1 exerted a protective role in the vasculature. Mechanically, NR1D1 deficiency aggravates macrophage infiltration, inflammation, and oxidative stress. Compared with the ApoE−/− mice, NR1D1−/−ApoE−/− mice exhibited a significantly higher expression level of pyroptosis-related genes in macrophages within the plaque. Further investigation based on mice bone marrow-derived macrophages (BMDMs) confirmed that NR1D1 exerted a protective effect by inhibiting macrophage pyroptosis in a NLRP3-inflammasome-dependent manner. Besides, pharmacological activation of NR1D1 by SR9009, a specific NR1D1 agonist, prevented plaque vulnerability/rupture. In general, our findings provide further evidences that NR1D1 plays a protective role in the vasculature, regulates inflammation and oxidative stress, and stabilizes rupture-prone vulnerable plaques.


Introduction
Atherosclerosis, characterized by the formation of plaques containing lipids, cells, debris, and scar tissue in the arterial walls, causes much morbidity and mortality worldwide [1,2]. While atherosclerosis is a chronic inflammation induced by cholesterol, inflammation could alter vascular function and contribute to the atherosclerotic plaque progression in a lipidindependent manner [3]. Rupture at the site of a vulnerable atherosclerotic plaque is the most frequent cause of acute coronary syndrome, but mechanisms responsible for plaque vulnerability and rupture remain largely unknown [4]. It is well known that inflammation contributes to the vulnerability of plaques which are the substrate of acute coronary syndrome [5]. Recent studies demonstrated that macrophage pyroptosis initiated by inflammation conversely augmented the inflammatory response in the vasculature and contributed to the rupture of vulnerable plaques [6,7].
Pyroptosis is an inflammatory form of programmed cell death characterized by the formation of gasdermin-(GSDM-) based pores on the plasma membrane, which is triggered by the binding of damage/danger-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) by inflammasomes and pattern recognition receptors (PRR) [8,9]. Canonical pathways initiate from the activation of the inflammasome complex (NLRP3, NLRP1, NAIP-NLRC4, AIM2, and Pyrin), which provides a platform for the maturation of caspase-1. Caspase-1 leads to the activation of interleukin-(IL-) 1β and IL-18, as well as gasdermin D (GSDMD). Activated inflammasome induces formation of cytoplasmic membrane pores, swelling, and rapid cell rupture and thus releases proinflammatory factors and cell contents. Of interest, mounting evidences suggested that pyroptosis of macrophages within atherosclerotic plaques may contribute to plaque vulnerability/rupture [7,10]. Hence, a more specific understanding of mechanisms underlying macrophage pyroptosis in vulnerable plaques is urgent and may provide a novel therapeutic target for the rupture-prone vulnerable plaques and consequent cardiovascular events.
Nuclear receptor subfamily 1 group D member 1 (NR1D1), also known as REV-ERB α, is a member of the nuclear receptor superfamily, functioning as a transcriptional repressor [11]. NR1D1 and other circadian clock proteins (RORs, BMAL, CLOCK, PER, and CRY) are involved in many physiological processes, including regulation of metabolism, development, immunity, and circadian rhythm [12]. Interestingly, emerging evidence suggests that NR1D1 exerts a powerful anti-inflammatory and antioxidative stress effect [13]. Recent studies identified its protective role in various cardiovascular diseases including myocardial infarction and cardiac hypertrophy [14][15][16].
By using loss-of-function approach (global NR1D1 −/− mice) and pharmacological activation of NR1D1 by SR9009, we aimed at determining the effect and molecular mechanisms of NR1D1 on atherosclerotic plaque vulnerability/rupture and the potential involvement of NR1D1 in mediating the vasoprotective effects.

Animal Models of Rupture-Prone Vulnerable Plaques.
Animal experiments were referred to the National Institutes of Health Guidelines on the Use of Laboratory Animals and followed the institutional guidelines outlined by the Animal Ethics Committee of Shanghai Jiao Tong University. NR1D1-deficient (NR1D1 −/− ) mice on a C57BL/6 background were kindly given by Dr. Ueli Schibler (University of Geneva, Geneva, Switzerland) [17]. The NR1D1 −/− mice were bred with ApoE −/− mice to establish NR1D1 −/− mice under an ApoE −/− background. A total of 23 NR1D1 −/ − ApoE −/− male mice and 69 ApoE −/− male mice at 8 weeks of age were maintained for subsequent study.
For animal models of rupture-prone vulnerable plaques, we used a previously published protocol [18]. In brief, eightweek-old male mice were allowed to acclimate for a week prior to the study and were fed a high-fat diet (1.25% cholesterol and 21% fat; 42% kcal as fat) during the experiment. The rupture-prone vulnerable carotid plaques were induced by partly ligating the left renal artery and the left internal and the external carotid arteries by using perivascular collars. Plaque rupture was defined by an area of fibrous cap disruption. Intraplaque hemorrhage is defined as the deposition of blood products inside the plaque and is not necessarily associated with atherosclerotic plaque rupture. All the mice underwent general anesthesia using pentobarbi-tal sodium at a dose of 100 mg/kg and were maintained on a heating pad. To study the effect of SR9009 on plaque vulnerability/rupture, mice after surgery were randomly divided into the vehicle group (phosphate buffer saline (PBS)) and the SR9009-treated group (100 mg/kg/d, MCE, cat.no. HY-16989, dissolved in PBS and given by intraperitoneal injection once a day) [19,20].

Microultrasound
Imaging. At baseline and postoperative weeks 4 and 8, microultrasound images were obtained with a 55 MHz transducer via the Vevo 770 system (VisualSonics) under isoflurane (2%) inhalation anesthesia. Prior to the ultrasound examination, the hair around the necks of the mice was shaved for better image quality. Standard techniques were used to obtain B-mode measurements of the length of the atherosclerotic lesion in the left common carotid artery and extreme values of intimal-medial thickness (IMT). Additionally, the maximal flow velocity (V max) proximal to the stenosis was obtained in a pulse-wave Doppler mode. To measure the degree of eccentric plaque distribution, the eccentric index (EI) was calculated from the extreme values of IMT by the formula ðmaximal IMT − minimal IMTÞ/maximal IMT.

Tissue Collection and Analysis.
At postoperative week 8, mice were sacrificed by deep anesthesia for tissue collection after measuring blood pressure and body weight. Plasma was obtained from the blood samples of mice by centrifugation at 1000 g for 15 minutes at 4°C and was stored at −80°C. After perfusion, the specimens of the carotid arteries were obtained and kept in paraffin or embedded in OCT compound (Sakura Finetek USA Inc., Torrance, CA, United States).
2.4. Histopathology and Immunofluorescence/Immunohistochemistry. The carotid arteries specimens were embedded in the OCT compound and serially sectioned (5 μm). Hematoxylin and eosin (H&E), Masson's trichrome, and Sirius red staining were performed according to the manufacturers' protocols (Sigma). To evaluate the lipid content in the vasculature, sections were stained with oil red O, using Mayer's hematoxylin (Sigma) as the counter stain.
For immunofluorescence analysis, serial paraffin sections were incubated with different primary antibodies overnight at 4°C, followed by the corresponding fluorochrome-conjugated antibodies for detection. The following primary antibodies were used: rat monoclonal antibody against CD68 (Abcam, ab53444), rabbit monoclonal antibody against NLRP3 (Abcam, ab270449), rabbit monoclonal antibody against IL-1β (Abcam, ab234437), rabbit polyclonal antibody against caspase-1 (ABclonal, A16792), and rabbit monoclonal antibody against NR1D1 (Abcam, ab174309). After they reacted with the corresponding Alexa Fluor 488 or 594-conjugated secondary antibodies (Invitrogen), DAPI was added to visualize nuclei. 2.6. BMDM Culture and Treatment. Eight-to ten-week-old C57/B6 mice were euthanized according to the animal protocol approved by the Animal Ethics Committee of Shanghai Jiao Tong University. Femurs were flushed with PBS, and mononuclear phagocyte progenitor cells from the bone marrow were collected in the medium. The medium was centrifuged at 1000 rpm for 5 minutes. After removing the supernatant, the precipitate was incubated with red cell lysate at 4°C for 5 minutes and centrifuged again. The cells were plated at the density of 2 × 10 6 /mL in six-well culture plates and cultured in DMEM supplemented with 10% FBS and 1% P/S and 20 ng/mL M-CSF (PeproTech). Culture media were replaced on day 3 and day 6. On day 7, BMDMs were administrated with ox-LDL (50 μg/mL) and SR9009 (10 μmol/L) [20][21][22].

Real-Time Quantitative PCR.
Total RNA was extracted from carotid arteries or BMDMs using TRIzol Reagent (Invitrogen). cDNA was synthesized using RT SuperMix for qPCR (Vazyme) and then amplified by SYBR qPCR master mix (Vazyme) in the LightCycler® 480 Real-Time PCR System (Roche). The primer sequences utilized for real-time PCR are presented in Table S1. Results from LightCycler 480 analysis software are presented as Ct values, normalized against GAPDH, and shown as 2 −ΔΔCt .
2.9. Oxidative Stress Detection In Vivo and In Vitro. Reactive oxygen species (ROS) generation in the vasculature was measured by the dihydroethidium (DHE) staining. Briefly, serial frozen sections of carotid arteries (5 μm) were incubated with DHE (5 μmol/L) in a light-protected humidified chamber at room temperature for 60 min. After incubation, DAPI was added to visualize the nuclei. Fluorescent images were acquired with a laser scanning confocal microscope (Leica TCS SP8, Leica Microsystems). All the images and staining intensities were acquired and measured with Image-Pro Plus 6.0 (Media Cybernetics Inc.). For detecting reactive oxygen species (ROS) generation in BMDMs, 12-well plates precoated with Matrigel solution (BD) were stored at 4°C overnight. BMDMs were seeded on the precoated plates and incubated at 37°C and 5% CO 2 . After administration of ox-LDL and SR9009, BMDMs were fixed with 4% paraformaldehyde. After being permeabilized in Triton X-100 and blocked in PBS containing 10% BSA, BMDMs were successively stained with 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (DCFH-DA) (10 μM, Sigma) for 30 minutes at room temperature. After incubation, DAPI was added to visualize the nuclei. Cells were examined with a laser scanning confocal microscope (Leica TCS SP8, Leica Microsystems). The percentage of DCFH-DA-positive cells was analyzed with Image-Pro Plus 6.0 (Media Cybernetics Inc.).

Terminal Deoxynucleotidyl Transferase dUTP Nick End
Labelling (TUNEL) Assay. DNA damage of macrophages in carotid plaque lesions was detected by the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). Serially sectioned paraffin carotid artery specimens (5 μm) were incubated with anti-CD68 primary antibodies (Abcam, ab53444). Sections were then incubated with TUNEL reaction mixture according to the manufacturer's protocol. After incubation, DAPI was added to visualize the nuclei.
To detect DNA damage of BMDMs after administration of ox-LDL and SR9009, BMDMs were fixed with 4% paraformaldehyde. After being permeabilized in Triton X-100 and blocked in PBS containing 10% BSA, BMDMs were successively stained with TUNEL reaction mixture according to the manufacturer's protocol. After incubation, DAPI was added to visualize the nuclei. Images were acquired with a laser-scanning confocal microscope (Leica TCS SP8, Leica Microsystems). All the images and staining intensities were acquired and measured with Image-Pro Plus 6.0 (Media Cybernetics Inc.).   Oxidative Medicine and Cellular Longevity using IBM-SPSS v.24.0 statistical software (IBM) as the mean ± SEM and analyzed with one-sided t test for two independent samples or one-way ANOVA followed by post hoc Tukey's test for sets of data. Categorical variables were compared using chi-square analysis. P values were calculated with a 95% confidence level.

NR1D1 Expression Is downregulated during Vulnerable
Plaque Formation. To explore whether NR1D1 is involved in the pathological progression of vulnerable plaque, we first investigated the biological roles of NR1D1 in rupture-prone 5 Oxidative Medicine and Cellular Longevity vulnerable carotid plaques. Both immunofluorescence staining of plaque and tissue RT-q-PCR showed that NR1D1 expression levels were significantly reduced in vulnerable carotid plaques (Figures 1(a)-1(c)), suggesting that NR1D1 may exert protective effects in the vasculature. We further explored the expression change of NR1D1 in macrophages, endothelial cells, and vascular smooth muscle cells after ox-LDL administration. NR1D1 expression was not changed in endothelial cells and vascular smooth muscle cells, while it was found to be significantly downregulated after ox-LDL stimulation in macrophages (Figures 1(d)-1(f)). The altered NR1D1 expression in rupture-prone vulnerable carotid plaques implied a potential protective role of NR1D1 in vulnerable plaque progression.

NR1D1 Deficiency Aggravates Macrophage Infiltration
and Inflammation in the Vasculature. It is reported that NR1D1 could exert anti-inflammatory effects in various diseases [23][24][25]. We further determined the effects of NR1D1 deficiency on inflammation and macrophage infiltration in the vasculature. Immunofluorescence staining of CD68 showed that macrophage infiltration occurred in vulnerable plaques, which was dramatically augmented by NR1D1 deficiency (Figure 3(a)). Moreover, immunofluorescence staining of plaque revealed that NR1D1 deficiency significantly upregulated NF-κB P65 expression (Figure 3 (Figure 3(d)). These data suggested that NR1D1 deficiency significantly aggravated the macrophage infiltration and inflammation in carotid artery lesions.

NR1D1 Deficiency Aggravates Oxidative Stress in the
Vasculature. We further evaluated the roles of NR1D1 in regulating oxidative stress in vulnerable plaques. DHE staining indicated that NR1D1 deficiency significantly increased generation of ROS in vulnerable plaques (Figure 4(a)). We further assessed the effects of NR1D1 deficiency on genes related to oxidative stress. The results of tissue RT-q-PCR revealed that several antioxidative genes including CAT, SOD1, and GPX4 were significantly downregulated while NOX2 was upregulated in NR1D1 −/− ApoE −/− mice (Figure 4(b)). Consistent with that, the levels of oxidative stress-related indicators in serum samples were also significantly increased in NR1D1 −/− ApoE −/− mice (Figure 4(c)). Collectively, these data suggested that NR1D1 deficiency greatly increased oxidative stress generation in carotid artery lesions.  Macrophage pyroptosis contributes to plaque vulnerability and promotes plaque rupture [5]. NR1D1 is known to regulate genes involved in pyroptosis [22]. Thus, we further determined the effects of NR1D1 deficiency on macrophage pyroptosis. Interestingly, we observed a significant increase of TUNEL + macrophages in carotid plaque lesions in NR1D1 −/− ApoE −/− mice ( Figure 5(a)). Double immunofluorescence staining indicated that macrophages in carotid plaque lesions in NR1D1 −/− ApoE −/− mice exhibited increased expression of pyroptosis-related genes such as NLRP3, IL-1β, and caspase-1 (Figures 5(b)-5(d)) compared to ApoE −/− mice. Taken together, our results suggested that NR1D1 deficiency activated NLRP3 inflammasome, upregulated IL-1β and caspase-1, and thus induced macrophage pyroptosis and rupture-prone vulnerable plaques.
3.6. SR9009 Treatment Mitigates ox-LDL-Induced Inflammation, Oxidative Stress, and BMDM Pyroptosis. To explore the potential protective effects of NR1D1 in stabilizing rupture-prone plaques, we first determined the effects of SR9009 (a specific NR1D1 agonist) treatment on ox-LDL-stimulated BMDMs. Consistent with the in vivo study, ox-LDL stimulation decreased the expression of NR1D1 in BMDMs (Figure 6(a)). Meanwhile, Western blotting showed that SR9009 decreased the phosphorylation of NF-κB P65 (Figure 6(a)), which indicated an antiinflammatory effect of SR9009. DCFH-DA assay indicated that SR9009 treatment reduced the ROS generation (Figure 6(b)).
The NLRP3 inflammasome has been identified as a critical regulatory factor in macrophage pyroptosis [26]. It has been demonstrated that NR1D1 regulates the expression of NLRP3 through manipulating transcription. Thus, we further investigated the effects of SR9009 on inflammasome priming. Consistent with the in vivo study, SR9009 treatment significantly reduced TUNEL + BMDMs (Figure 6(c)). Moreover, Western blotting showed that SR9009 treatment significantly inhibited ox-LDL-induced pyroptosis-related gene expression (NLRP3, GSDMD, IL-1β, and caspase-1) (Figure 6(d)). These observations indicated that upregulation of NR1D1 by SR9009 inhibited macrophage pyroptosis and ROS generation through the NLRP3 inflammasome pathway.
3.7. SR9009 Treatment Improves Stability of Rupture-Prone Vulnerable Plaques. We further determined the protective effects of NR1D1 in vivo. SR9009 treatment significantly reduced the incidence of intraplaque hemorrhage (13.04% vs. 43.48%, P = 0:0472) and spontaneous plaque rupture with thrombus (8.70% vs. 37.78%, P = 0:0706) and thus improved the stability of rupture-prone vulnerable plaque (Figure 7(a)). Moreover, functional analysis with microultrasound imaging verified the protective effects of SR9009. Decreased IMT, V max , and EI were observed in SR9009-treated mice (Figure 7(b)). The atherosclerotic area, lipid content, and collagen/lipid ratio were also significantly decreased in SR9009treated mice (Figures 7(c) and 7(d)). Consistent with the  Figure S1). These data suggested that SR9009 treatment stabilized rupture-prone vulnerable plaques.

Discussion
In the present study, we first investigated the role of the nuclear receptor NR1D1 in the vulnerable plaques and demonstrated the protective effects of NR1D1 in the vasculature. By establishing a rupture-prone vulnerable plaque model, we demonstrated that NR1D1 deficiency significantly increased the incidence of intraplaque hemorrhage and spontaneous plaque rupture with the thrombus. Inflammation and oxidative stress were aggravated in NR1D1 −/− ApoE −/− mice; thus, macrophage pyroptosis was induced. A further study in BMDMs identified the role of NR1D1 in manipulating macrophage pyroptosis by regulating NLRP3 inflammasome. Taken together, our study provided new insights into the significant roles of NR1D1 in the vasculature and suggested that NR1D1 was a unique endogenous protective nuclear receptor against rupture-prone vulnerable plaques by regulating inflammatory response, ROS generation, and macrophage pyroptosis.
Nuclear receptors (NRs) are widely involved in various essential biological functions such as cell growth and death, embryonic development, cellular metabolism, immune response, and inflammation [27,28]. Mounting evidences suggest that NRs play a pivotal role in maintaining cardio-vascular homeostasis, while deficiency of NRs contributes to a wide array of diseases such as atherosclerosis, abdominal aortic aneurysm, and ischemic cardiac injury [18,29,30]. NR1D1, a unique member of the nuclear receptor super family, is well known for regulating the circadian rhythm, immune response, and metabolism [31,32]. Numerous studies have proved that NR1D1 exerted a protective effect by regulating energy metabolism and inhibiting inflammation [33,34]. Ma et al. found that LDLR −/− mice receiving NR1D1 knockdown bone marrow transplantation developed greater atherosclerotic plaques [35]. Sitaula et al. found that the administration of SR9009 suppressed the progression of atherosclerosis [19]. They both identified that NR1D1 regulated the polarization of macrophages and inhibited inflammation in the vasculature during the atherosclerosis progression. These studies focused on the role of NR1D1 in the progression of atherosclerotic plaque in a mouse model induced with high-fat diet. The majority of cardiovascular events are caused by rupture of vulnerable atherosclerotic plaques [36][37][38]. At present, there is a general understanding of the initiation and progression of atherosclerosis but the process of the transition from stable plaque to vulnerable plaque remains to be elucidated [2,39]. Although progress have been made in treating atherosclerosis of the early stage, the effective strategies targeting vulnerable plaques are still lacking [40]. It is of great importance to understand the mechanisms of plaque vulnerability and rupture and to explore the potential drug to stabilize rupture-prone vulnerable plaques. We established the particular rupture-prone vulnerable plaque model to explore the underlying mechanism of plaque vulnerability/rupture. Consistent with former studies, we found that NR1D1 exerted a protective role in the vasculature. Furthermore, we first identified the deficiency of NR1D1-augmented atherosclerotic plaque vulnerability with higher incidence of intraplaque hemorrhage and plaque rupture with thrombus. Targeting NR1D1 by SR9009 significantly prevented the atherosclerotic plaque vulnerability and rupture. In general, our results extended upon the established vasoprotective roles of NR1D1 during atherogenesis and identified SR9009 as a novel drug in stabilizing the rupture-prone vulnerable plaques. Macrophages play important roles in all stages of atherosclerosis, from the initiation of lesions and lesion expansion, to necrosis leading to rupture and multiple complications [41,42]. The status of macrophages rather than absolute infiltration number contributes to the inflammation and oxidative stress in the vasculature, leading to plaque vulnerability and rupture [43,44]. Within the plaques, macrophage pyroptosis induces robust inflammatory response, releases proinflammatory cytokines such as IL-1β and IL-18, and thus initiates a deteriorating process causing increased inflammation and oxidative stress [45][46][47]. Together, these events promote the formation of a necrotic core and thus the vulnerability of the rupture-prone plaques [48]. In addition to macrophages, endothelial cells, smooth muscle cells, etc. also contribute to the progression of atherosclerotic plaques [2]. Whereas macrophage is now considered to play an essential role in the initiation, progression, and especially the final steps of atherosclerosis, namely, plaque vulnerability and eventually plaque rupture [49,50]. Besides, in vitro experiments showed that ox-LDL stimulation did not alter the expression level of NR1D1 in smooth muscle cells and endothelial cell, while the expression level of NR1D1 in macrophages was significantly downregulated, indicating that NR1D1 mainly manipulates the plaque vulnerability via regulating macrophages.
Inflammasomes including the nucleotide-binding oligomerization domain-(NOD-) like receptor (NLR) family (NLRP3, NLRP1, NLRC4, NLRP9, and NLRP6) and PYHIN protein families (absent in melanoma 2 (AIM2)) detect DAMPs and trigger the canonical pyroptosis signaling pathway [51][52][53]. Specifically, it has been verified that the NLRP3-caspase-1 signaling pathway is vital in mediating macrophage pyroptosis [54,55]. DAMPs including inflammatory cytokines and cholesterol crystals activate the NLRP3 inflammasome to bind to adaptor protein apoptosis-associated speck-like protein containing CARD domain (ASC), which recruits procaspase-1. The recruited procaspase-1 is catalytically activated to generate caspase-1 [56,57]. Active caspase-1 is responsible for processing and maturing IL-1β/18 and cleaving the GSDMD to induce macrophage pyroptosis [58]. Indeed, recent studies demonstrated that NR1D1 could regulate the transcription of NLRP3 thus mediating the pyroptosis signaling pathway [20,22]. We observed that the plaque vulnerability was significantly increased in NR1D1 −/− ApoE −/− mice, accompanied with increased macrophage pyroptosis in the vasculature. Activation of NR1D1 by SR9009 significantly inhibited macrophage pyroptosis and prevented plaque vulnerability/rupture. In general, our results demonstrated that NR1D1 might manipulate macrophage pyroptosis in a NLRP3-caspase-1-dependent man-ner within the vulnerable plaques. Recent developments of NR1D1 pharmacology suggest that this nuclear receptor is a druggable target and that ligands targeting NR1D1 may be useful in the treatment of several diseases [12]. Various studies suggest that SR9009, the most common NR1D1 agonist, significantly inhibits inflammation and regulates metabolism and immune response via upregulating NR1D1 [59][60][61]. Moreover, our results broaden the application fields of SR9009, provide new perspectives for the treatment of vulnerable plaques, and may promote the clinical application of SR9009.
In summary, NR1D1 deficiency aggravated inflammation and oxidative stress in the vasculature and thus induced macrophage pyroptosis and rupture-prone vulnerable plaques. Targeting NR1D1 by SR9009 may represent a novel protective strategy for stabilizing rupture-prone vulnerable plaques.

Data Availability
Data are available on request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.