Abstract Background Atherosclerosis is a progressive arterial disease characterized by chronic inflammation and plaque formation in blood vessel walls. ELABELA, an endogenous ligand for the G protein‐coupled receptor APJ (apelin peptide jejunum, apelin receptor), has multiple pharmacological activities for protecting the cardiovascular system. This study aimed to determine the potential antiatherosclerotic effect of ELABELA and reveal the underlying mechanisms. Methods We enrolled a cohort consisting of patients with and without atherosclerosis to determine the relationship between plasma ELABELA levels and atherosclerosis severity. The potential therapeutic action of ELA‐21 (ELABELA‐21) on atherosclerosis in high‐fat diet‐fed ApoE ^−/− mice was evaluated. Results Plasma ELABELA levels were significantly reduced and negatively correlated with plasma MMP2 (matrix metallopeptidase 2) and MMP9 (matrix metallopeptidase 9) levels in patients with atherosclerosis and high‐fat diet‐induced atherosclerotic ApoE ^−/− mice. Plasma ELABELA levels exhibited a potential diagnostic value for patients with atherosclerosis. Applying ELA‐21 significantly decreased the atherosclerotic plaque area and inflammation in the aortas of the ApoE ^−/− mice. ELA‐21 administration modulated the balance between M1 and M2 macrophages in the abdominal cavity and aorta roots toward a more anti‐inflammatory status, accompanied by reduced MMP2, MMP9, and PRR ([pro]renin receptor), and enhanced macrophage APJ, ACE (angiotensin‐converting enzyme), and ACE2 (angiotensin‐converting enzyme 2) protein expression in plaques within aortic roots and decreased plasma soluble PRR levels. In vitro, ELA‐21 effectively suppressed oxidized low‐density lipoprotein‐induced foam cell formation and lipopolysaccharide/interferon‐γ‐induced M1 polarization in cultured macrophages. Interestingly, the anti‐inflammatory effect of ELA‐21 was further enhanced by APJ inhibitor ML221 [4‐oxo‐6‐((pyrimidin‐2‐ylthio)methyl)‐4H‐pyran‐3‐yl 4‐nitrobenzoate], accompanied by elevated ACE and ATP6AP2 (ATPase, H^+‐transporting, lysosomal accessory protein 2) and reduced ACE2 mRNA levels. Conclusions Our data highlighted the diagnostic and therapeutic potential of ELABELA on atherosclerosis. ELA‐21 protects against atherosclerosis by inhibiting atherosclerotic plaque formation and promoting a more stable plaque phenotype, possibly via restoring the M1/M2 macrophage balance, enhancing macrophage ACE and ACE2 expression, and inhibiting the PRR system. ELABELA may be a novel diagnostic biomarker and candidate therapeutic target for atherosclerosis. Keywords: atherosclerosis, ELABELA, macrophage, renin‐angiotensin system Subject Categories: Atherosclerosis, Biomarkers, Basic Science Research, ACE/Angiotension Receptors/Renin Angiotensin System, Inflammation __________________________________________________________________ Nonstandard Abbreviations and Acronyms α‐SMA α‐smooth muscle actin ACE2 angiotensin‐converting enzyme 2 APJ apelin peptide jejunum CD68 cluster of differentiation 68 EC endothelial cell ELA‐21 ELABELA‐21 HFD high‐fat diet IFN‐γ interferon‐γ iNOS inducible nitric oxide synthase MMP2/9 matrix metallopeptidase 2/9 ox‐LDL oxidized low‐density lipoprotein PRR (pro)renin receptor RAS renin‐angiotensin system sPRR soluble (pro)renin receptor TIMP4 tissue inhibitor of metalloproteinases 4 VSMC vascular smooth muscle cell Research Perspective. What Is New? * In patients with atherosclerosis, reduced plasma ELABELA levels correlate negatively with disease severity, suggesting its diagnostic potential for atherosclerosis. * ELA‐21 (ELABELA‐21) protects against atherosclerosis by restoring M1/M2 macrophage balance, upregulating ACE (angiotensin‐converting enzyme) and ACE2 (angiotensin‐converting enzyme 2) expression, and inhibiting the PRR ([pro]renin receptor) system, highlighting its therapeutic potential for atherosclerosis. What Question Should Be Addressed Next? * The specific receptor mediating ELA‐21's antiatherosclerotic effect, the impact of macrophage‐specific ELABELA/APJ (apelin peptide jejunum) on atherosclerosis, and how ELA‐21 stimulates macrophage ACE and ACE2 should be clarified. Atherosclerosis, a chronic inflammatory disease, is not only the leading cause of the morbidity and mortality of cardiovascular diseases globally but also the primary pathological basis of most cardiovascular diseases.[48] ^1 Atherosclerosis is initiated by endothelial dysfunction, followed by the adhesion of monocytes; monocytes differentiate into macrophages under the stimulation of macrophage‐stimulating factor and other cytokines and the formation of foam cells after the engulfment of oxidized low‐density lipoprotein (ox‐LDL) by macrophages, the release of contents from the dead foam cells, and gradual aggregation of vascular smooth muscle cells (VSMCs), collagen, and foam cells, eventually forming atherosclerotic plaque.[49] ^2 Currently, the first‐line drugs widely used in clinical practice for atherosclerosis are mainly lipid‐lowering drugs, such as statins (inhibiting [3‐hydroxy‐3‐methyl glutaryl coenzyme A] reductase), fibrates, and preprotein converting enzyme subtilisin 9 inhibitors.[50] ^3 In addition, antiplatelet drugs, such as aspirin[51] ^4 and clopidogrel,[52] ^5 and anti‐inflammatory drugs, such as renin‐angiotensin system (RAS) inhibitors,[53] ^6 are also commonly used in clinical practice for atherosclerosis. However, although the above drugs have slowed down atherosclerosis progression to a certain extent, their efficacy in reducing the mortality of cardiovascular diseases is only about 30%. They are accompanied by potential adverse reactions. For instance, ezetimibe can cause respiratory infection, muscle pain, and joint pain, and RAS inhibitors can cause hyperkalemia. Therefore, novel, innovative, and safer pharmacological interventions and antiatherosclerotic drugs are needed to ameliorate atherosclerotic cardiovascular diseases and thus mortality worldwide. The apelineargic system, an important endocrine system in the body, consists of 2 endogenous peptide ligands, apelin (encoded by Apln) and ELABELA (ELA; encoded by Apela and also known as Toddler), as well as the G protein‐coupled receptor APJ (Apelin peptide jejunum, Apelin receptor, encoded by Aplnr).[54] ^7 In human atherosclerotic plaques, apelin and APJ are colocalized with α‐SMA (α‐smooth muscle actin) in VSMCs and CD68 (cluster of differentiation 68) in macrophages.[55] ^8 Although upregulated apelin and APJ levels were observed in the human atherosclerotic coronary artery,[56] ^8 increasing studies have reported decreased plasma apelin levels in patients with acute coronary syndrome,[57] ^9 , [58]^10 rheumatoid arthritis,[59] ^11 and symptomatic intracranial atherosclerotic stenosis.[60] ^12 In particular, plasma apelin levels were positively associated with plasma MMP2 (matrix metallopeptidase 2) levels but negatively correlated with plasma MMP9 (matrix metallopeptidase 9) levels in patients with rheumatoid arthritis,[61] ^11 implying a positive correlation between plasma apelin levels and atherosclerotic plaque stability. This notion was supported by the results observed in patients with acute coronary syndrome that plasma apelin levels were significantly lower in patients with the ruptured plaque than in those with the nonruptured plaque and inversely associated with plaque cross‐sectional area but positively related with external elastic membrane cross‐sectional area.[62] ^9 Apelin can not only inhibit the formation of foam cells by activating cell autophagy and inhibiting lipid accumulation in macrophages but also promote M2 polarization of macrophages, thereby inhibiting the release of proinflammatory factors,[63] ^13 , [64]^14 , [65]^15 implying the potential therapeutic action of apelin on atherosclerosis. Apelin‐13 has been reported to inhibit angiotensin II‐induced atherosclerosis in ApoE ^−/− mice by increasing NO bioavailability.[66] ^16 Similarly, apelin‐13 also significantly enhanced the stability of atherosclerotic plaque by increasing the collagen content in the plaque and reducing the expression of MMP9, the infiltration of inflammatory cells (neutrophils and macrophages), and the levels of reactive oxygen species in the plaque, without affecting the lesion size in high‐fat diet (HFD)‐induced atherosclerotic ApoE ^−/− mice.[67] ^17 Overall, these results suggest the important clinical value of apelin in diagnosing and treating atherosclerosis. Of note, Hashimoto et al have reported that systemic APJ knockout significantly reduced atherosclerotic lesions by inhibiting oxidative stress in VSMCs and cell proliferation without affecting cholesterol levels in high‐cholesterol‐fed ApoE ^−/− mice.[68] ^18 These results may imply that the regulatory effect of APJ on atherosclerosis seems to be opposite to that of apelin, or the antiatherosclerotic effects of apelin may be independent of APJ. Similar to the bioeffects of apelin, ELA also displays significant protective actions on the cardiovascular system.[69] ^7 A study has demonstrated a decreased circulating ELA level in patients with hypertension with an increased carotid intima‐media thickness (IMT),[70] ^19 implying a potential negative correlation between circulating ELA and subclinical atherosclerosis. However, the clinical significance of ELA in patients with atherosclerosis and the impact of ELA on atherosclerosis progression remain unclear. The aims of this study are: (1) to determine plasma ELA levels in patients with atherosclerosis and assess the correlation between plasma ELA and atherosclerosis severity and (2) to evaluate the potential therapeutic effect of ELA‐21 (ELABELA‐21) on atherosclerosis in HFD‐fed ApoE ^−/− mice. METHODS Data Availability Statement The authors declare that all supporting data are available within the article and its online supplementary files. The raw data supporting the conclusions of this article will be made available by Dr Xu without undue reservation. Ethics Statement The studies involving human participants were reviewed and approved by the Affiliated Hospital of the Jiangxi University of Chinese Medicine (JZFYLL20230208002). The patients/participants provided written informed consent to participate in this study. The studies involving animals were reviewed and approved by the Animal Care and Use Committee, which also approved the animal protocols at Jiangxi University of Chinese Medicine (number JZLLSC20230254). Study Population The study protocol was approved by the ethics committee of the Affiliated Hospital of the Jiangxi University of Chinese Medicine (JZFYLL20230208002) and performed under the Declaration of Helsinki ethical principles and STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) cohort reporting guidelines.[71] ^20 All participants provided written informed consent and clinical characteristics after a full explanation of the purpose of the study and the potential risk involved. The inclusion and exclusion criteria for enrolled patients with atherosclerosis and the collection method of plasma samples were previously described in detail.[72] ^21 Briefly, the diagnosis of subclinical atherosclerosis was established after evaluating the carotid IMT and plaque area according to the bilateral carotid ultrasonography examination; patients with heart diseases (heart failure, rheumatic heart disease, valvular heart disease, and cardiomyopathy), hepatic failure, kidney disease (chronic and acute kidney injury), hyperthyroidism, malignancy, pulmonary embolism, and other inflammatory diseases were excluded. Blood samples were collected from 236 patients/participants between December 2022 and December 2023 at the Department of Cardiology, Affiliated Hospital of Jiangxi University of Chinese Medicine. All fasting blood samples were collected from a peripheral vein of all patients within 24 hours of admission and immediately centrifuged for 5 minutes at 4 °C and 1000 x g to separate plasma, which was immediately stored at −80 °C until use. None of the patients/participants received the optimized treatment before collecting blood samples. The participants were divided into nonatherosclerosis and atherosclerosis groups. All laboratory assessments, except plasma ELA and apelin levels, were conducted in the clinical laboratory center of the Affiliated Hospital of the Jiangxi University of Chinese Medicine according to the standard protocols. High‐Fat‐Induced Atherosclerosis in ApoE ^−/− Mice Male and female ApoE ^−/− mice (6–8 weeks old) were purchased from Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China. Mice were all given free access to tap water and the standard diet. Mice were housed in individually ventilated cages (temperature 20–26 °C, humidity 40%–70%), with a 12:12‐hour light–dark cycle. The Animal Care and Use Committee approved the animal protocols at the Jiangxi University of Chinese Medicine (number JZLLSC20230254), and all processes were in strict accordance with the National Institutes of Health Guide for the Care and Use of Animals in laboratory experiments and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.[73] ^22 All mice received HFD (kcal%: 41% fat, 43% carbohydrate, 17% protein) (number H10141; Beijing Hfk Bioscience, Beijing, China) feeding for 12 weeks and then were randomly divided into 2 groups; mice in the HFD group received intraperitoneal injection of the vehicle (0.9% physiological saline), and mice in the HFD + ELA‐21 group received intraperitoneal injection of ELA‐21 (LRKHNCLQRRCMPLHSRVPFP, >98% purity; GenScript) in 0.9% physiological saline (1 mg/kg per day, 3 times a day, every 8 hours) for an additional 4 weeks, based on and modified from previous studies.[74] ^23 , [75]^24 At the end of the experiments, mice were anesthetized by isoflurane, and then plasma, peritoneal macrophages, full‐length aorta, and the aortic root were harvested for further analysis. En Face Oil‐Red‐O Staining of Full‐Length Aortas The fat‐stripped full‐length aortas of ApoE ^−/− mice were fixed at 4 °C in 4% paraformaldehyde/0.1 M phosphate salt buffer and dehydrated with a 30% sucrose gradient. The aortas were longitudinally cut open to fully expose the inner surface, followed by 2 equilibration steps in 60% isopropanol for 5 minutes each and stained with 0.3% Oil‐Red‐O staining solution at room temperature for 20 minutes in the dark. The aortas were differentiated with 60% isopropanol 2 to 3 times and rinsed with distilled water to terminate the differentiation and remove the excess dye. Images were captured, and the atherosclerotic lesion areas were measured using Image‐Pro Plus version 6.0 software. Histological Examination and Immunofluorescence Staining of Aortic Root Lesions The aortic roots were fixed overnight at 4 °C in 4% paraformaldehyde/0.1 M phosphate salt buffer and dehydrated with a 30% sucrose gradient. The samples were embedded in an optimal cutting temperature compound and frozen using liquid nitrogen. Cryosections measuring 10 μm were obtained from the aortic root to the apex. Slides were used for hematoxylin and eosin, Oil‐Red‐O, and Masson staining for histological examination according to the manufacturers' instructions. Antibodies used for immunofluorescence staining are shown in Table [76]S1. Images were captured using a Leica DMI4000B fluorescence microscope (Wetzlar, Germany). The quantification of the sizes of both atherosclerotic plaque and collagen fibers was analyzed using Image‐Pro Plus version 6.0 software, and the average fluorescence intensity was quantified by calculating the total area of fluorescence intensity relative to the plaque area. Plasma Biochemical Analysis The plasma collected was instantaneously centrifuged at 1000 x g for 10 minutes at 4 °C. The plasma concentrations of ELA (S‐1508; Peninsula Laboratories), apelin (for human sample: E01T0015; Bluegene Tech, Shanghai, China; for mouse sample: EKE‐057‐15; Phoenix Pharmaceuticals, Burlingame, CA), and soluble PRR [(pro)renin receptor] (sPRR) (27 782; Immuno‐Biological Laboratories, Gunma, Japan) were evaluated by commercial ELISA kits according to the manufacturer's instructions, respectively. ELISA measurements of plasma MMP2 (HM10737 for human, MU30640 for mouse), MMP9 (HM10095 for human, MU30613 for mouse), IL (interleukin)‐1β (MU30369), IL‐6 (MU30044), TNF‐α (tumor necrosis factor‐α) (MU30030), and IL‐10 (MU30055) were performed following the manufacturer's instructions (Bioswamp, Wuhan, China). Transcriptome Sequencing and Data Processing Aortic samples in HFD and HFD + ELA‐21 groups (n = 3 for each group) were immediately harvested for total RNA extraction, and RNA quality was estimated to construct the transcriptomics sequencing library. Transcriptome sequencing and analysis were performed by Novogene Biotechnology (Beijing, China) using an Illumina Novaseq platform. Reads from all samples were mapped to the GRCm39 reference genome using the HISAT2 package (HISAT 2.2.1). Differential expression analysis between the 2 groups was conducted using the DESeq2 software (version 3.19). In this work, |log2FoldChange| ≥1.5 and Padj <0.05 were set as the threshold for defining differentially expressed genes. All differentially expressed genes were initially mapped to gene ontology (GO) terms in the GO database to identify Top10 enriched GO terms among the differentially expressed genes with a significance threshold of P < 0.05. Pathway enrichment analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify Top10 enriched signal transduction pathways among the differentially expressed genes with an adjusted P value <0.05. Cell Culture The THP‐1 (Tohoku Hospital Pediatrics‐1, a human myeloid leukemia mononuclear cell line) and RAW264.7 (a murine monocytic macrophage leukemia cell line) cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C with 5% CO[2]. They were treated with 100 ng/mL phorbol myristate acetate for 24 hours and then pretreated with ELA‐21 (0.1, 1, or 5 μM) for 12 hours, followed by LPS (lipopolysaccharide)/IFN‐γ (interferon‐γ) or ox‐LDL treatment for 24 hours. The expression of inflammatory cytokines or the formation of foam cells was detected. L929 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, and the supernatant of the culture media was collected as the L929 conditioned medium. Bone marrow cells were isolated from femurs and tibias of 8‐week‐old male C57BL/6 mice and cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 U/mL) for 72 hours. The floating cells were harvested and cultured in L929 conditioned medium. Bone marrow‐derived macrophages (BMDMs) were pretreated with ELA‐21 (0.1 or 1 μM) for 12 hours, followed by LPS/IFN‐γ treatment for 24 hours, and the expression of inflammatory cytokines was detected. Reverse Transcriptase‐Quantitative Polymerase Chain Reaction Total RNA was extracted from aortic samples, peritoneal macrophages, THP‐1 cells, RAW264.7 cells, and BMDMs using Trizol reagent (15 596 018; Life Technologies, Carlsbad, CA) and was reversed transcribed to cDNA using the HiScript Q RT SuperMix (R122; Vazyme, Nanjing, China). Real‐time reverse transcriptase‐quantitative polymerase chain reaction (RT‐qPCR) was performed using the Hieff qPCR SYBR Green Master Mix reagent (11201ES; Yeasen, Shanghai, China) and specific primer (Table [77]S2) in the Light Cycler 96 System (Roche, Ricardo Rojas, Argentina). Relative mRNA expression of each gene was shown as a relative value normalized by 18s or GAPDH. Statistical Analysis Continuous data in the cohort study were expressed as mean±SD for normally distributed data, median and interquartile range for nonnormally distributed data, and number and percentage for categorical variables. Data in the animal study were summarized as mean±SD. An unpaired Student t test was performed for statistical analysis for 2 comparisons using IBM SPSS 19 software. Spearman correlation analysis was used to correlate plasma ELA or apelin levels and study variables. The diagnostic value of plasma ELA and apelin was assessed by determining the area under the receiver operating characteristic curves (AUC) using IBM SPSS 19 software. All tests were 2‐sided, and P < 0.05 was considered statistically significant. RESULTS Plasma ELABELA and Apelin Levels in Patients With and Without Atherosclerosis The baseline characteristics of enrolled patients without atherosclerosis (n = 63, aged 58.8±11.7 years) and patients with atherosclerosis (n = 173, aged 67.1±11.0 years, P < 0.001 versus the nonatherosclerosis group) have been described in detail in our previous studies.[78] ^21 Briefly, there were no significant differences in sex, body mass index, comorbidities, or blood pressure between the 2 groups. Laboratory examinations revealed that plasma brain natriuretic peptide, urea nitrogen, and triglyceride levels were slightly higher, and plasma MMP2 (4.4 ± 1.8 versus 3.5 ± 1.9 ng/mL, P < 0.001) and MMP9 (2.7 ± 1.2 versus 1.7 ± 0.9 ng/mL, P < 0.001) levels were significantly higher in the atherosclerosis group than those in the nonatherosclerosis group. In contrast, other parameters, including plasma creatine, uric acid, low‐density lipoprotein cholesterol, high‐density lipoprotein‐cholesterol, and total cholesterol, were not different between the 2 groups. According to the echocardiographic data, the left and right carotid maximum IMT were significantly increased in the atherosclerosis group compared with those in the nonatherosclerosis group (P < 0.001). In contrast, other parameters reflecting cardiac function, including the left ventricular ejection fraction, are lower. The left atrial diameter, left ventricular end‐diastolic diameter, left ventricular end‐systolic diameter, left ventricular posterior wall thickness, interventricular septum thickness, and right ventricular internal dimension diameter were not significantly different between the 2 groups. The levels of plasma ELA in patients with atherosclerosis were substantially lower than those in the nonatherosclerosis group (9.4 ± 3.7 versus 14.9 ± 3.3 ng/mL, P < 0.001; Figure [79]1A). The mean plasma apelin (34.3 ± 31.1 versus 57.9 ± 56.0 ng/mL, P < 0.001; Figure [80]1B) levels of the atherosclerosis groups were also significantly lower than those in the nonatherosclerosis group. Figure 1. Plasma ELABELA and apelin levels in patients with AS. Figure 1 [81]Open in a new tab A and B, Plasma ELABELA (A) and apelin (B) levels assessed by ELISA assays. N = 63 for the non‐AS group and n = 173 for the AS group. ***P < 0.001 vs non‐AS. C, The correlation between plasma ELABELA and apelin. D and E, The correlation between plasma ELABELA and MMP2 (D) and MMP9 (E). F and G, The correlation between plasma apelin and MMP2 (F) and MMP9 (G). H and I, Receiver operating characteristic curves of plasma ELABELA, apelin, MMP2, and MMP9 levels (H) as well as their combinations (I) for AS. AS indicates atherosclerosis; MMP2, matrix metalloproteinase 2; and MMP9, matrix metalloproteinase 9. We analyzed the correlation between plasma ELA or apelin and study variables in all subjects (Table [82]S3). There was no correlation between plasma ELA and plasma apelin (r = 0.091, P = 0.165; Figure [83]1C). Age (r = −0.257, P < 0.001), plasma MMP2 levels (r = −0.221, P = 0.001; Figure [84]1D), plasma MMP9 levels (r = −0.280, P < 0.001; Figure [85]1E), left atrial diameter (r = −0.495, P < 0.001), left ventricular end‐systolic diameter (r = −0.490, P < 0.001), left ventricular posterior wall (r = −0.172, P = 0.008), interventricular septum thickness (r = −0.538, P < 0.001), left carotid maximum IMT (r = −0.307, P < 0.001), right carotid maximum IMT (r = −0.284, P < 0.001), left carotid plaque area (r = −0.330, P < 0.001), and left carotid plaque area (r = −0.311, P < 0.001) were negatively related to plasma ELA levels. Heart rate (r = 0.182, P = 0.005), plasma high‐density lipoprotein cholesterol levels (r = 0.146, P = 0.025), and left ventricular end‐diastolic diameter (r = 0.560, P < 0.001) were positively correlated to plasma ELA levels. In contrast, although there was no correlation between plasma apelin and plasma MMP2 levels (r = −0.036, P = 0.587; Figure [86]1F), plasma MMP9 levels (r = −0.132, P = 0.043; Figure [87]1G), left atrial diameter (r = −0.232, P < 0.001), left ventricular end‐systolic diameter (r = −0.223, P = 0.001), interventricular septum thickness (r = −0.243, P < 0.001), left carotid maximum IMT (r = −0.317, P < 0.001), right carotid maximum IMT (r = −0.245, P < 0.001), and left carotid plaque area (r = −0.146, P = 0.024) were negatively related to plasma apelin levels. Plasma triglyceride levels (r = 0.147, P = 0.024) and left ventricular end‐diastolic diameter (r = 0.282, P < 0.001) were positively correlated to plasma apelin levels. Multiple linear regression analyses in all participants were further performed to determine the relationship between plasma ELA levels, plasma apelin levels, and clinical characteristics associated with atherosclerosis (Table [88]S4). We found a significant association between plasma ELA levels and heart rate (β = 0.109, t = 2.877, P = 0.004), left atrial diameter (β = 0.217, t = 3.392, P = 0.001), left carotid maximum IMT (β = 7.980, t = 0.910, P = 0.046), and right carotid maximum IMT (β = −9.535, t = −1.143, P = 0.014). In contrast, plasma apelin levels were only associated with left carotid maximum IMT (β = −10.363, t = −3.268, P = 0.001) and right carotid maximum IMT (β = 6.206, t = 2.611, P = 0.010). To estimate the diagnostic values of ELA, apelin, MMP2/9, and their combinations for atherosclerosis, the AUC was analyzed for data from all patients with and without atherosclerosis (Figure [89]1H and [90]1I, Table [91]S5), followed by a pairwise comparison of receiver operating characteristic curves via the DeLong test (Table [92]S6). When a single diagnosis was used, ELA had the highest diagnostic value (AUC 0.855±0.025 [95% CI, 0.803–0.897], sensitivity 56.65%, specificity 96.83%), followed by MMP9 (AUC 0.803±0.036 [95% CI, 0.746–0.851], sensitivity 97.69%, specificity 52.38%), suggesting a potential diagnostic value of plasma ELA levels for atherosclerosis with moderate sensitivity and high specificity. When multiple joint diagnoses were performed, ELA combined with MMP9 had the highest diagnostic value (AUC 0.919±0.018 [95% CI, 0.877–0.951], sensitivity 74.57%, specificity 93.65%), followed by ELA + MMP2 (AUC 0.866±0.025 [95% CI, 0.816–0.907], sensitivity 90.17%, specificity 63.49%), and apelin+MMP9 (AUC 0.802±0.036 [95% CI, 0.745–0.851], sensitivity 100.00%, specificity 50.79%). Thus, a combined assessment of ELA and MMP2/MMP9 may be a good choice to increase the accuracy of the diagnosis of atherosclerosis. Plasma ELABELA and Apelin Levels in ApoE ^−/− Mice Fed an HFD To further assess the status of plasma ELA and apelin levels in the setting of atherosclerosis, the ApoE ^−/− mice were fed an HFD (42% fat) for 16 weeks to induce atherosclerosis, and then the levels of plasma ELA and apelin were examined in these mice. After HFD feeding, both the levels of plasma ELA (5.7 ± 3.6 versus 17.3 ± 3.2 ng/mL, P < 0.001; Figure [93]2A) and apelin (3.3 ± 0.9 versus 5.0 ± 0.6 ng/mL, P < 0.001; Figure [94]2B) were significantly reduced. A significant upregulation of plasma MMP2 (9.7 ± 1.1 versus 6.3 ± 1.1 ng/mL, P < 0.001) and MMP9 (4.7 ± 0.9 versus 2.3 ± 0.4 ng/mL, P < 0.001) levels were observed in these mice.[95] ^21 We also analyzed the correlation between plasma ELA, apelin, and plasma MMPs in HFD‐induced atherosclerotic mice. Plasma ELA levels were significantly positively correlated with plasma apelin (r = 0.607, P < 0.001) (Figure [96]2C) and negatively correlated with plasma MMP2 (r = −0.647, P < 0.001) (Figure [97]2D) and MMP9 levels (r = −0.627, P < 0.001) (Figure [98]2E) in these mice. A similar negative correlation between plasma apelin levels and plasma MMP2 (r = −0.602, P < 0.001) (Figure [99]2F) and MMP9 levels (r = −0.619, P < 0.001) (Figure [100]2G) was also observed. Figure 2. Plasma ELABELA and apelin levels in HFD‐fed ApoE ^−/− mice. Figure 2 [101]Open in a new tab A and B, Plasma ELABELA (A) and apelin (B) levels assessed by ELISA assays. N = 20 for the control group and n = 25 for the HFD group. ***P < 0.001 vs control. C, The correlation between plasma ELABELA and apelin. D and E, The correlation between plasma ELABELA and MMP2 (D) and MMP9 (E). F and G, The correlation between plasma apelin and MMP2 (F) and MMP9 (G). HFD indicates high‐fat diet; MMP2, matrix metalloproteinase 2; and MMP9, matrix metalloproteinase 9. ELA‐21 Attenuated Atherosclerosis in ApoE ^−/− Mice To study the impact of ELA‐21 on atherosclerosis, ApoE ^−/− mice were fed an HFD for 12 weeks, followed by a 4‐week ELA‐21 intervention. The technology roadmap of this study is shown in Figure [102]3A. Compared with the HFD group, ELA‐21 significantly reduced the lesion area, as reflected by the reduced lipid deposition assessed by Oil‐Red‐O staining in full‐length aortas of male (Figure [103]3B and [104]3C) and female (Figure [105]S1) ApoE ^−/− mice. To further clarify the effect of ELA‐21 on atherosclerotic plaques, Oil‐Red‐O staining, hematoxylin and eosin staining, and Masson's trichrome staining were performed to evaluate lipid deposition, necrotic core size, plaque area, and collagen content within the aortic roots, respectively. Compared with male HFD mice, there was a significant decrease in lipid deposition within the aortic root plaques in male HFD + ELA‐21 mice, accompanied by a significant reduction in the necrotic core size and plaque area within the aortic roots (Figure [106]3D). The lipid deposition within the aortic root plaques and plaque area within the aortic roots were also reduced by ELA‐21 treatment in female ApoE ^−/− mice (Figure [107]S2). Furthermore, the collagen fiber content within the aortic roots significantly increased in male HFD mice with ELA‐21 treatment. By immunofluorescence staining, although there was no difference in the macrophage content (CD68‐positive cells) in the aortic root between the 2 groups, the α‐SMA‐positive area was significantly increased in the male HFD + ELA‐21 group (Figure [108]3E). These results collectively indicate a significant therapeutic action of ELA‐21 on atherosclerosis by preventing the progression of atherosclerotic lesions and promoting a more stable plaque phenotype. Figure 3. ELA‐21 attenuated atherosclerosis in male ApoE ^−/− mice induced by an HFD. Figure 3 [109]Open in a new tab A, Experimental procedure for HFD‐induced atherosclerosis in male ApoE ^−/− mice and ELA‐21 administration. B, Oil‐Red‐O staining of the full‐length aorta from the male ApoE ^−/− mice. C, Quantification of the plaques in the full‐length aorta, aortic arch, thoracic aorta, and abdominal aorta. D, Masson, Oil‐Red O, and H&E staining of the aortic root from the male ApoE ^−/− mice, and the collagen fiber content, intraplaque fat content, plaque area, and necrotic lesion area in the aortic root plaque were quantified. E, CD68 and α‐SMA immunofluorescence staining in the aortic root from the male ApoE ^−/− mice (×200) and the positive staining of CD68 and α‐SMA in the aortic root plaque was quantified. Data are mean±SEM. *P < 0.05, **P < 0.01. α‐SMA indicates α‐smooth muscle actin; CD68, cluster of differentiation 68; ELA‐21, ELABELA‐21; H&E, hematoxylin and eosin; HFD, high‐fat diet; and ORO, Oil‐Red O. ELA‐21 Suppressed Inflammation and the Formation of Foam Cells of Macrophages It is well accepted that inflammation plays a significant role in the progression of atherosclerosis, especially the process of plaque rupture.[110] ^1 We conducted immunofluorescence staining to examine the distribution of expression of endogenous ELA within the aortic root plaques in ApoE ^−/− mice. As shown in Figure [111]S3, ELA was mainly colocalized with CD68 but not α‐SMA, confirming the predominant localization in macrophages within the aortic root plaques. We also detected the mRNA expression levels of inflammatory cytokines in the full‐length aortas and peritoneal macrophages from the ApoE ^−/− mice by RT‐qPCR. ELA‐21 treatment significantly reduced aortic Tnf‐α, Il‐1β, Il‐12b, Inos, Il‐6, and monocyte chemoattractant protein‐1 mRNA expression in HFD‐fed male ApoE ^−/− mice (Figure [112]4A). The mRNA levels of Tnf‐α, Il‐1β, Inos, Il‐6, and monocyte chemoattractant protein‐1 in the full‐length aortas of HFD‐fed female ApoE ^−/− mice were also significantly decreased after ELA‐21 treatment (Figure [113]S4). Macrophages, which can polarize toward proinflammatory M1 or anti‐inflammatory M2 phenotypes, play a central role in atherosclerotic lesions.[114] ^25 In this study, the macrophage infiltration within the plaque area in the aortic roots from HFD‐fed male ApoE ^−/− mice, as reflected by CD68 immunofluorescence staining, was not affected by ELA‐21 (Figure [115]3E). However, EAL‐21 reduced the percentage of iNOS (inducible nitric oxide synthase)^+CD68^+ cells while increasing the portion of Arg1 (arginase 1)^+CD68^+ cells within the aortic root plaques as reflected by the colabeling of the anti‐CD68 antibody with the anti‐iNOS antibody (Figure [116]4B) or the anti‐Arg1 antibody (Figure [117]4C). In peritoneal macrophages from HFD‐fed male ApoE ^−/− mice, ELA‐21 significantly reduced the mRNA expression of Il‐1β, Tnf‐α, Inos, and Il‐12β (the markers of M1 macrophages) while elevating the mRNA levels of Fizz1, Il‐10, and Cd206 (cluster of differentiation 206) (the markers of M2 macrophages) (Figure [118]4D). Thus, ELA‐21 treatment modulated the balance between M1 and M2 macrophages toward a more anti‐inflammatory status in HFD‐fed ApoE ^−/− mice. In addition, ELA‐21 significantly decreased plasma levels of IL‐1β, TNF‐α, and IL‐6 but increased plasma levels of IL‐6 in HFD‐fed male ApoE ^−/− mice (Figure [119]4E). Figure 4. ELA‐21 suppressed inflammation in male ApoE ^−/− mice induced by an HFD. Figure 4 [120]Open in a new tab A, RT‐qPCR represents the expression of inflammatory factors in the full‐length aorta from the male ApoE ^−/− mice. B and C, iNOS (B) and Arg1 (C) immunofluorescence staining in the aortic root from the male ApoE ^−/− mice (×200) and the positive staining of iNOS and Arg1 in CD68‐positive area was quantified. Scale bar = 100 μm. D, RT‐qPCR represents the expression of inflammatory factors in peritoneal macrophages from the male ApoE ^−/− mice. E, Plasma levels of inflammatory factors in the ApoE ^−/− mice assessed by ELISA. Data are mean±SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Arg1 indicates arginase 1; CD68, cluster of differentiation 68; DAPI, 4′,6‐diamidino‐2‐phenylindole; ELA‐21, ELABELA‐21; HFD, high‐fat diet; iNOS, inducible nitric oxide synthase; and RT‐qPCR, reverse transcriptase‐quantitative polymerase chain reaction. To further clarify the effect of ELA‐21 on M1 polarization, ELA‐21 intervention was used in LPS/IFN‐γ‐treated THP‐1 and RAW264.7 macrophages and mouse BMDMs in vitro, and RT‐qPCR analysis was performed to detect the specific markers of M1 macrophages. In RAW264.7 cells, LPS/IFN‐γ‐stimulated Tnf‐α, Il‐1β, Inos, and Il‐12b mRNA expression was consistently attenuated by ELA‐21 treatment at least in high‐doses (5 μM) (Figure [121]5A). The same experiments that were performed using the THP‐1 cells yielded similar results. Administration of ELA‐21 significantly abolished LPS/IFN‐γ‐induced upregulation of TNF‐α, IL‐1β, IL‐6, iNOS, and IL‐12b mRNA levels in the THP‐1 cells (Figure [122]5B). Similarly, in mouse BMDMs, ELA‐21 also significantly attenuated LPS/IFN‐γ‐stimulated Tnf‐α, Il‐1β, Il‐6, and Il‐12b mRNA expression (Figure [123]5C). Collectively, these results consistently suggest the inhibitory effect of ELA‐21 on the M1 polarization of macrophages, which may at least in part contribute to the improved atherosclerotic lesions. To further verify whether the anti‐inflammatory action of ELA‐21 depends on APJ in the macrophages, an APJ‐specific inhibitor ML221 [4‐oxo‐6‐((pyrimidin‐2‐ylthio)methyl)‐4H‐pyran‐3‐yl 4‐nitrobenzoate] was used. Interestingly, ELA‐21‐downregulated TNF‐α and IL‐6 mRNA expression in THP‐1 cells was further decreased by ML221 treatment (Figure [124]5D). These results suggest that the inhibitory effect of ELA‐21 on macrophage inflammation may depend on an unknown receptor independent of the APJ. Figure 5. ELA‐21 abolished the M1 polarization of macrophages in vitro. Figure 5 [125]Open in a new tab Mouse RAW264.7 (a murine monocytic macrophage leukemia cell line)(A), human THP‐1 (a human myeloid leukemia mononuclear cell line)(B and D) macrophages, and mouse BMDMs (C) were stimulated by LPS/IFN‐γ for 24 hours to induce M1 polarization in the presence or absence of ELA‐21 or an APJ inhibitor ML221, RT‐qPCR was performed to detect the mRNA levels of M1 polarization‐related markers in macrophages. *P < 0.05, **P < 0.01, ***P < 0.001, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001, ^$$$ P < 0.001. APJ indicates apelin peptide jejunum; BMDMs, bone marrow‐derived macrophages; ELA‐21, ELABELA‐21; IFN‐γ, interferon‐γ; LPS, lipopolysaccharide; ML221, 4‐oxo‐6‐((pyrimidin‐2‐ylthio)methyl)‐4H‐pyran‐3‐yl 4‐nitrobenzoate; RT‐qPCR, reverse transcriptase‐quantitative polymerase chain reaction; and THP‐1, Tohoku Hospital Pediatrics‐1. Macrophage foaming, one of the main hallmarks of early atherosclerosis lesions, is caused by the accumulation of cholesterol esters due to the excessive uptake of ox‐LDL.[126] ^26 Foam cell formation and accumulation are hallmarks of atherosclerosis progression and are new targets for fighting atherosclerosis.[127] ^26 Here, we also investigated the possible impact of ELA‐21 on macrophage foam cell formation in vivo and in vitro. First, HFD‐caused lipid deposition in peritoneal macrophages from the male ApoE ^−/− mice assessed by Oil‐Red‐O staining was significantly abolished by ELA‐21 administration (Figure [128]S5A). Second, ELA‐21 significantly attenuated ox‐LDL incubation‐induced lipid deposition in cultured THP‐1 macrophages assessed by Oil‐Red‐O staining (Figure [129]S5B). These results indicate an inhibitory action of ELA‐21 on macrophage foaming, which may also contribute to its antiatherosclerotic effect. ELA‐21 Enhanced Timp4 Expression and Reduced MMP Levels To further clarify the potential mechanisms of ELA‐21's antiatherosclerotic action, transcriptome sequencing of full‐length aortas from HFD‐fed ApoE ^−/− mice was performed. As shown in Figure [130]6A through [131]6C, 160 genes were upregulated, and 153 genes were downregulated by ELA‐21 administration. Among them, the 5 genes with the most significant increased expression levels include FK506‐binding protein 5 (Fkbp5), tissue inhibitor of metalloproteinases 4 (Timp4), alkaline ceramidase 2 (Acer2)‌‌, retinol binding protein 7 (Rbp7), and zinc finger and BTB domain containing 16 (Zbtb16), whereas the 5 genes with the most significant decreased levels include hyaluronan synthase 1 (Has1), small ubiquitin related modifier protein 2 (Sumo2), nuclear receptor subfamily 1, group D, member 1 (Nr1d1), B‐cell CLL/lymphoma 3 (Bcl3), and Il‐6 (Figure [132]6B). The regulated genes were significantly enriched in lipid and atherosclerosis‐related pathways assessed by GO and KEGG enrichment analysis (Figure [133]6C). The imbalance between MMPs and their inhibitors (TIMPs [tissue inhibitor of metalloproteinase]) leads to the imbalance of proteolytic activity and adverse extracellular matrix remodeling, which is closely related to the initiation, progression, and rupture of atherosclerotic plaques.[134] ^27 , [135]^28 We thus detected the levels of MMPs and TIMPs in HFD‐fed ApoE ^−/− mice to clarify whether ELA‐21 plays an antiatherosclerotic role by regulating the MMPs and TIMPs. By RT‐qPCR, administration of ELA‐21 significantly decreased Timp1, Timp2, Timp3, Mmp2, and Mmp9 mRNA levels but enhanced Timp4 mRNA expression in the full‐length aorta from HFD‐fed ApoE ^−/− mice (Figure [136]6D). We also conducted immunofluorescence staining to examine the expression of MMP2 and MMP9 in the aortic roots. The sections were colabeled with anti‐CD68 antibody. The signal within the plaque from anti‐MMP2 antibody (Figure [137]6E) and anti‐MMP9 antibody (Figure [138]6F) was significantly weakened by ELA‐21, indicating the reduced MMP2 and MMP9 protein expression in the aortic roots after ELA‐21 treatment. Furthermore, we compared plasma MMP2 and MMP9 levels between HFD and HFD + ELA‐21 groups using ELISA. The plasma levels of MMP2 and MMP9 in HFD‐fed ApoE ^−/− mice were significantly reduced by ELA‐21 administration (Figure [139]6G). To further clarify the regulation of ELA‐21 on MMP2 and MMP9 expression, we also detected the mRNA levels of MMP2 and MMP9 in cultured THP‐1 cells stimulated by LPS/IFN‐γ, in which ELA‐21 treatment significantly decreased both MMP2 and MMP9 mRNA levels (Figure [140]6H). Thus, ELA‐21 may enhance the stability of plaques by stimulating TIMP4 expression and inhibiting the release of MMPs. Figure 6. Regulation of ELA‐21 on the expression of TIMPs and MMPs in the aorta from the HFD‐fed male ApoE ^−/− mice. Figure 6 [141]Open in a new tab A through C, mRNA sequencing results of the full‐length aorta from the HFD‐fed male ApoE ^−/− mice. A, Cluster diagram of differentially expressed genes. B, Volcanic diagram of differentially expressed genes. C, GO and KEGG enrichment plot of differentially expressed genes. D, RT‐qPCR results of Timps and Mmps in the aorta from the HFD‐fed male ApoE ^−/− mice. E and F, MMP2 (E) and MMP9 (F) immunofluorescence staining in the aortic root from the HFD‐fed male ApoE ^−/− mice (×200) and the positive staining of MMP2 and MMP9 in CD68‐positive area was quantified. G, Plasma MMP2 and MMP9 concentrations in the male ApoE ^−/− mice assessed by ELISA. H, RT‐qPCR results of MMP2 and MMP9 mRNA in the LPS/IFN‐γ‐treated THP‐1 cells in the presence or absence of ELA‐21. *P < 0.05, **P < 0.01, ***P < 0.001, ^# P < 0.05, ^### P < 0.001. CD68 indicates cluster of differentiation 68; DAPI, 4′,6‐diamidino‐2‐phenylindole; ELA‐21, ELABELA‐21; GO, Gene ontology; HFD, high‐fat diet; IFN‐γ, interferon‐γ; IL, interleukin; KEGG, Kyoto Encyclopedia of Genes and Genomes; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; MMP2, matrix metalloproteinase 2; MMP9, matrix metalloproteinase 9; RT‐qPCR, reverse transcriptase‐quantitative polymerase chain reaction; TIMP, tissue inhibitor of metalloproteinases; THP‐1, Tohoku Hospital Pediatrics‐1, a human myeloid leukemia mononuclear cell line; and TNF, tumor necrosis factor. Modulation of ELA‐21 on the Macrophage RAS There is a close correlation between atherosclerotic plaque formation and the RAS, an important regulator of the inflammatory response.[142] ^6 , [143]^29 To clarify the regulation of ELA‐21 on the RAS in macrophages in the setting of atherosclerosis, we conducted immunofluorescence staining to examine the expression of APJ, angiotensin‐converting enzyme (ACE), ACE2 (angiotensin converting enzyme 2), and PRR in the aortic roots, which were colabeled with anti‐CD68 antibody. EAL‐21 significantly increased the percentage of APJ^+CD68^+ (Figure [144]7A), ACE^+CD68^+ (Figure [145]7B), and ACE2^+CD68^+ (Figure [146]7C) cells while reducing the portion of PRR^+CD68^+ (Figure [147]7D) cells within the aortic root plaques. These results indicate the stimulatory role of ELA‐21 on APJ, ACE, and ACE2 protein expression and the inhibitory effect of ELA‐21 on PRR protein expression in macrophages under atherosclerotic conditions. Plasma sPRR levels in HFD‐fed ApoE^−/− mice were significantly decreased by ELA‐21 (Figure [148]7E). We also used RT‐qPCR to detect Aplnr, Ace, Ace2, and Atp6ap2 (ATPase, H^+‐transporting, lysosomal accessory protein 2) mRNA levels in the full‐length aorta from HFD‐fed ApoE ^−/− mice. As shown in Figure [149]7F, administration of ELA‐21 significantly reduced Ace mRNA levels but elevated Ace2 mRNA levels in the full‐length aorta from HFD‐fed ApoE ^−/− mice without affecting Aplnr and Atp6ap2 mRNA levels. Furthermore, although LPS/IFN‐γ stimulated both ACE and ACE2 mRNA expression and reduced ATP6AP2 mRNA levels, ELA‐21 only further elevated ACE2 mRNA levels in cultured THP‐1 cells (Figure [150]7F). Interestingly, ML221 treatment further enhanced ACE and ATP6AP2 mRNA expression but abolished ELA‐21‐upregulated ACE2 mRNA levels (Figure [151]7G). Thus, the stimulation of ELA‐21 on ACE2 expression may depend on APJ, whereas APJ may be required to inhibit ACE and PRR expression in macrophages. Figure 7. Regulation of ELA‐21 on the expression of APJ, ACE, ACE2, and PRR in the aortic root from the HFD‐fed male ApoE ^−/− mice. Figure 7 [152]Open in a new tab A through D, APJ (A), ACE (B), ACE2 (C), and PRR (D) immunofluorescence staining in the aortic root from the HFD‐fed male ApoE ^−/− mice (×200) and the positive staining of APJ, ACE, ACE2, and PRR in CD68‐positive area was quantified. E, Plasma sPRR concentrations in the HFD‐fed male ApoE ^−/− mice assessed by ELISA. F, RT‐qPCR represents the expression of Aplnr, Ace, Ace2, and Atp6ap2 mRNA in the full‐length aorta from the HFD‐fed male ApoE ^−/− mice. G, RT‐qPCR represents the expression of ACE, ACE2, and ATP6AP2 mRNA in LPS/IFN‐γ‐treated THP‐1 cells with and without ELA‐21 or ML221 (an APJ inhibitor) treatment. *P < 0.05, **P < 0.01, ^### P < 0.001, ^$ P < 0.05, ^$$ P < 0.01. ACE indicates angiotensin‐converting enzyme; ACE2, angiotensin‐converting enzyme 2; APJ, apelin peptide jejunum; ATP6AP2, ATPase, H^+‐transporting, lysosomal accessory protein 2; CD68, cluster of differentiation 68; DAPI, 4′,6‐diamidino‐2‐phenylindole; ELA‐21, ELABELA‐21; HFD, high‐fat diet; IFN‐γ, interferon‐γ; LPS, lipopolysaccharide; ML221, 4‐oxo‐6‐((pyrimidin‐2‐ylthio)methyl)‐4H‐pyran‐3‐yl 4‐nitrobenzoate; PRR, (pro)renin receptor; RT‐qPCR, reverse transcriptase‐quantitative polymerase chain reaction; and sPRR, soluble (pro)renin receptor. DISCUSSION The current study examined the potential correlation between ELA and atherosclerosis progression. On the one hand, our data have demonstrated the diagnostic potential of ELA in atherosclerosis, as reflected by the significant reduction of plasma ELA levels and its significant diagnostic value in patients with atherosclerosis. Plasma ELA levels negatively correlated with atherosclerosis severity. On the other hand, our data have demonstrated the therapeutic potential of ELA‐21 on atherosclerosis, as reflected by the reduced lesion size within the aorta and enhanced stable atherosclerotic plaque phenotypes in HFD‐fed ApoE ^−/− mice with ELA‐21 intervention. Mechanistically, the antiatherosclerotic action of ELA‐21 may be associated with the restored balance of the M1/M2 macrophage, enhanced expression of ACE and ACE2 in macrophages, and inhibited PRR system. Our findings may provide valuable insights for using ELA in patients with atherosclerosis and call for clinical evaluation of its antiatherosclerotic efficacy and diagnostic effectiveness in individuals with atherosclerotic conditions. However, clinical therapeutic use of peptide drugs is mainly limited by the short in vivo half‐life, and parenteral administration of these peptides, including ELA‐21,[153] ^30 novel modifications to improve the in vivo stability of ELA‐21, and new delivery methods to improve the efficiency of ELA‐21 binding specific receptors are encouraged to develop. In the present study, we systematically studied the potential relationship between ELA and atherosclerosis and provided direct evidence for the potential diagnostic and therapeutic role of ELA in atherosclerosis. First, in patients with atherosclerosis and atherosclerotic ApoE ^−/− mice, plasma ELA levels were significantly decreased and inversely related to plasma MMP2 and MMP9, 2 important biomarkers of atherosclerotic plaque instability,[154] ^28 implying a possible association between plasma ELA levels and plaque vulnerability. Decreased plasma ELA levels may create a tendency toward vulnerable plaque. Although Apela mRNA expression as assessed by RT‐qPCR appears to be more prevalent during development with high expression in the heart and kidney of embryonic rodents and restricted to renal tubular epithelial cells of adult rat and mouse kidneys,[155] ^23 , [156]^31 , [157]^32 other cells, such as endothelial cells, may also express ELA, because tamoxifen‐inducible endothelial‐specific deletion of ELA impaired perfusion recovery and angiogenesis in mice.[158] ^33 More importantly, we found that endogenous ELA is predominantly expressed in macrophages within atherosclerotic plaques. These may indicate the possible universal expression pattern of ELA, but the major resources of the plasma ELA are unclear and need to be clarified in the future. Second, plasma ELA exhibited a higher diagnostic value for atherosclerosis than plasma apelin. ELA combined with MMP2 or MMP9 may significantly elevate the diagnostic accuracy of atherosclerosis. Third, in HFD‐fed ApoE ^−/− mice, application of ELA‐21 not only significantly reduced lipid accumulation in the aortas and atherosclerotic plaque size in aortic roots but also increased collagen accumulation and α‐SMA (a marker of contractile VSMCs) expression in the aortic roots, accompanied with reduced MMP2 and MMP9 levels in the plasma and aortas and elevated aortic Timp4 mRNA expression. Of note, MMP2 and MMP9 mediate the stimulating effect of inflammatory cytokines on the migration and proliferation of VSMCs,[159] ^28 which themselves are central to maintaining the integrity and stability of the atherosclerotic plaque fibrous cap.[160] ^34 This contradicts the decrease of MMP2/MMP9 levels and the increase of α‐SMA levels within atherosclerotic plaques in the presence of ELA‐21. The different cell sources and functions of MMP2/MMP9 and α‐SMA during the process of plaque formation and progression may be used to explain the above contradiction. In this regard, MMP2 and MMP9 mainly come from macrophages and synthetic VSMCs, contributing to the increase of plaque fragility.[161] ^28 In contrast, α‐SMA is expressed by contractile VSMCs and maintains the stability of plaque structure.[162] ^35 Proinflammatory cytokines promote the transformation of VSMCs from contractile type to synthetic type, resulting in enhanced MMP2/MMP9 secretion and reduced α‐SMA expression.[163] ^35 Thus, the inhibition of inflammation by ELA‐21 may be the cause of the reduction of MMP2/MMP9 levels and elevation of α‐SMA levels, which directly reflects the inhibition of destructive inflammatory reaction and the enhancement of reparative smooth muscle response within atherosclerotic plaques. Our data suggest the potential of ELA‐21 to reduce the lesion size and enhance atherosclerotic plaque stability, but the feasibility and effectiveness of ELA‐21 in treating atherosclerosis need to be further clarified using an advanced atherosclerosis model and an atherosclerotic model with unstable plaque. Of note, APJ, the recognized receptor of ELA and apelin, exhibits an opposite regulatory effect on atherosclerosis to ELA and apelin on atherosclerosis.[164] ^18 These may indicate that the antiatherosclerotic action of apelin‐13 and ELA‐21 may be independent of the APJ. APJ‐independent actions of ELA via alternative, unknown cell‐surface receptors have already been reported in human embryonic stem cells[165] ^36 and renal cells,[166] ^23 , [167]^31 in which APJ is lacking. More relevantly, these unknown receptors, such as APJ, bind to the C‐terminus of ELA peptides.[168] ^37 , [169]^38 Thus, it is of great interest to identify the other alternative, unknown receptors for ELA, and understand the potential APJ‐independent mechanisms for an antiatherosclerotic role of ELA‐21. Interestingly, although the levels of the total APJ protein in aortic roots and Aplnr mRNA in the aortas from HFD‐fed ApoE ^−/− mice were unchanged, the APJ levels in CD68‐positive cells in plaques were significantly elevated upon ELA‐21 treatment. This may imply the possible antiatherosclerotic action of macrophage APJ, which may differ from that of APJ in other cell types such as VSMCs. However, the elevated macrophage APJ in plaques may also be a compensatory response to inhibiting inflammatory signals downstream of the APJ. Thus, the effect of APJ in macrophages versus in VSMCs on atherosclerosis progression needs further verification. In addition, it is important to clarify the direct overexpression of ELA‐21 peptide within the plaques to demonstrate the probable activation of ELA‐related signals. In this respect, fluorescent‐labeled ELA‐21 peptide in combination with ELA‐related receptor deficient mice, at least macrophage‐specific APJ‐deficient mice, may be helpful to address this issue. The M1 polarization of macrophage and subsequent foam cell formation and inflammatory response plays a central role in the atherosclerosis.[170] ^39 Apelin/APJ signaling plays an essential role in macrophage growth, survival, and physiological and pathological functions.[171] ^40 In particular, apelin has been reported to inhibit macrophage inflammation[172] ^15 , [173]^41 , [174]^42 and foam cell formation.[175] ^13 , [176]^14 Although several studies have demonstrated the anti‐inflammatory effect of ELA in VSMCs,[177] ^43 endothelial cells (ECs),[178] ^44 fibroblasts,[179] ^45 and peritoneal macrophages,[180] ^46 a report on the regulatory role of ELA on the polarization and foam cell formation of macrophages is still lacking. In the present study, ELA‐21 treatment did not affect the macrophage infiltration but significantly reduced iNOS^+CD68^+ macrophages and increased Arg1^+CD68^+ macrophages in the plaque in HFD‐induced atherosclerotic ApoE ^−/− mice. In addition, ELA‐21 significantly decreased the mRNA expression of the markers of M1 macrophages and elevated the mRNA levels of the markers of M2 macrophages in peritoneal macrophages from these mice. These results consistently indicate the modulation of the balance between M1 and M2 macrophages toward a more anti‐inflammatory status by ELA‐21, which may contribute to the improvement of atherosclerosis in HFD‐fed ApoE ^−/− mice. The inhibitory effect of ELA‐21 on M1 polarization was further replicated in in vitro cell experiments. In cultured THP‐1 cells, RAW264.7 cells, and mouse BMDMs, LPS/IFN‐γ‐induced M1 polarization was significantly abolished by ELA‐21 incubation. Interestingly, ELA‐21‐downregulated IL‐6 and TNF‐α mRNA expression were further reduced by an APJ‐specific inhibitor ML221 in LPS/IFN‐γ‐stimulated THP‐1 cells. These results suggest that the inhibitory effect of ELA‐21 on M1 polarization and inflammation may be independent of APJ, which may further support the APJ‐independent antiatherosclerotic action of ELA‐21 via unknown receptors. Also, ELA‐21 significantly attenuated the peritoneal macrophage foam cell formation from HFD‐fed ApoE ^−/− mice and ox‐LDL‐induced foam cell formation of THP‐1 cells. Collectively, our data highlighted the antiatherosclerotic effect of ELA‐21, possibly by restoring the M1/M2 macrophage balance and inhibiting foam cell formation of macrophages. However, the antiatherosclerotic impact and the specific downstream targets of ELA need to be further determined using mice with macrophage or EC‐specific ELA deficiency. Additionally, it should be noted that ELA‐21‐induced protective effects may be mediated through different cell types in the atherosclerotic aorta in addition to the macrophages, because receptors of ELA, including APJ, were universally distributed in multiple cell types.[181] ^47 Thus, single‐cell RNA sequencing for the isolated atherosclerotic aorta is recommended to determine the relative importance of ECs, VSMCs, versus macrophages for the antiatherosclerosis action of ELA‐21. The RAS, an important regulator of the inflammatory response, exhibits a close correlation with atherosclerotic plaque formation.[182] ^6 , [183]^29 ACE and ACE2 were present in both ECs, VSMCs, and macrophages in atherosclerotic lesions, but ACE was predominant in macrophages.[184] ^48 , [185]^49 Although the ACE inhibitor enalapril significantly inhibited AngI but not angiotensin II‐induced atherosclerosis in Ldlr ^−/− mice, the transplantation of macrophages lacking ACE into Ldlr ^−/− mice has no effect on the atherosclerotic lesion in aortic roots and only slightly reduce the lesion in aortic arch.[186] ^48 Similarly, vascular‐specific deletion of ACE (ACE3/3 mice, only expressing ACE in the liver and kidney)[187] ^50 or transplantation of macrophages lacking ACE into ApoE ^−/− mice[188] ^51 does also not affect the formation of atherosclerotic plaques in mice. However, a series of reports from the Bernstein group has shown that, although macrophage‐specific ACE overexpression (ACE10/10 mice) enhanced the inflammatory response of macrophages themselves, it significantly reduced atherosclerotic plaque by enhancing macrophage lipid metabolism, cholesterol efflux, and efferocytosis in HFD‐fed ApoE ^−/− mice[189] ^51 and AAV‐PCSK9 (adeno‐associated virus‐proprotein convertase subtilisin/kexin type 9)/HFD‐induced atherosclerotic mice.[190] ^52 , [191]^53 Apart from the macrophage ACE, EC‐specific deletion of ACE significantly reduced serum ACE activity without affecting the atherosclerotic lesion. In contrast, VSMC‐specific deletion of ACE significantly inhibited the atherosclerotic lesion without affecting the serum ACE activity in HFD‐fed Ldlr ^−/− mice.[192] ^54 Thus, there may be a cellular difference in the regulation of ACE on atherosclerosis that ACE in VSMCs rather than macrophages or ECs may be required for the occurrence and development of atherosclerosis, but enhanced macrophage ACE may exhibit a significant antiatherosclerotic action. In our study, although ELA‐21 significantly reduced Ace mRNA expression in the aortas, it significantly enhanced ACE protein expression in CD68‐positive cells in aortic roots from HFD‐fed ApoE ^−/− mice. However, although ACE mRNA expression in LPS/IFN‐γ‐stimulated THP‐1 cells was unaffected by ELA‐21, APJ inhibitor ML221 significantly elevated ACE mRNA levels. These results may indicate that ELA‐21 may stimulate macrophage ACE expression through other receptors independent of APJ, whereas APJ itself potentially inhibits ACE expression. However, due to the proinflammatory effect of macrophage ACE itself,[193] ^51 the increased expression of ACE in macrophages may be attributed to the reduced inflammatory response of macrophages. Overall, enhancement of macrophage ACE protein may contribute to the antiatherosclerotic effect of ELA‐21 by enhancing macrophage lipid metabolism and cholesterol efflux. Compared with the diverse effects of ACE on atherosclerosis, the role of ACE2 is much clearer and more consistent. Systemic ACE2 deletion significantly increased atherosclerotic plaques in ApoE ^−/− mice, accompanied by significantly increased expression of adhesion factors, inflammatory cytokines, and MMP9, and the proinflammatory response in macrophages, ECs, and VSMCs.[194] ^55 , [195]^56 Similarly, in HFD‐fed Ldlr ^−/− mice, ACE2 deletion in macrophages significantly increased atherosclerotic plaque and promoted the release of inflammatory cytokines and the adhesion of monocytes to ECs.[196] ^57 On the contrary, overexpression of ACE2 in monocytes or ECs leads to reduced endothelial adhesion and migration and the expression of adhesion factors.[197] ^58 , [198]^59 Recombinant adenovirus‐mediated ACE2 overexpression significantly reduced atherosclerotic plaques in HFD‐fed ApoE ^−/− mice[199] ^59 and enhanced atherosclerotic plaque stability in New Zealand rabbits by improving endothelial function, inhibiting inflammatory response and monocyte adhesion, reducing macrophage infiltration, and inhibiting the ERK (extracellular regulated protein kinase)/p38 (p38 MAP kinase), JAK (Janus Kinase)/STAT (Signal Transducer and Activator of Transcription), and NF‐κB (nuclear factor kappa‐B) signaling pathways.[200] ^60 More interestingly, in ApoE ^−/− mice with diabetes and ApoE ^−/− mice lacking ACE2, recombinant mouse soluble ACE2 protein treatment significantly reduced atherosclerotic plaque.[201] ^61 In addition, dimenazene, an activator of ACE2, significantly enhanced the stability of plaque by increasing the collagen content, reducing MMP9 expression and macrophage infiltration, and promoting M2 polarization of macrophages, accompanied by decreased expression of chemokines and inflammatory factors in plaques.[202] ^62 ACE2 overexpression inhibited M1 polarization of macrophages by suppressing the NF‐κB signaling pathway.[203] ^63 These results consistently showed an antiatherosclerotic role of ACE2 by inhibiting the formation and stability of atherosclerotic plaque. In the present study, the administration of ELA‐21 increased Ace2 mRNA levels in the aortas and elevated ACE2 protein levels in CD68‐positive cells in aortic roots from HFD‐fed ApoE ^−/− mice. In LPS/IFN‐γ‐stimulated THP‐1 cells, ACE2 mRNA expression was significantly enhanced by ELA‐21, which was abolished by APJ inhibitor ML221. These results suggest the stimulation of ELA‐21/APJ on ACE2 expression in both mRNA and protein levels in macrophages. The protective action of ELA on cell injury by stimulating ACE2 expression has already been reported in cardiomyocytes,[204] ^64 aortic fibroblasts,[205] ^45 and renal tubular cells.[206] ^31 Overall, stimulation of macrophage ACE2 may contribute to the antiatherosclerotic action of ELA‐21 by inhibiting M1 polarization and subsequent inflammatory response. The PRR system, a key regulator of the RAS, exhibits a potential correlation with atherosclerosis.[207] ^21 , [208]^65 , [209]^66 , [210]^67 Although the impact of PRR on atherosclerosis may exhibit species differences ([211]https://kns.cnki.net/KCMS/detail/detail.aspx?dbname=CMFD202002&fi lename=1019909467.nh),[212] ^66 macrophage‐specific PRR deletion significantly aggravated atherosclerosis by augmenting inflammation and impairing cholesterol efflux in HFD‐fed ApoE ^−/− mice, caused by vacuolar H^+‐ATPase (V‐ATPase) dysfunction rather than RAS activation.[213] ^66 These results indicate the possible antiatherosclerotic action of macrophage PRR by maintaining the activity of the V‐ATPase. However, elevated circulating sPRR may contribute to atherosclerosis progression through its proinflammatory effect by interacting and activating the AT1R (angiotensin type 1 receptor)[214] ^68 in macrophages. In this regard, both At1r mRNA levels in macrophages from the abdominal cavity[215] ^69 and plasma sPRR level[216] ^21 , [217]^70 , [218]^71 were elevated in mice with HFD feeding. AT1R inhibitor valsartan significantly reduced ox‐LDL and Il‐1β and Tnf‐α mRNA levels in macrophages.[219] ^69 Additionally, elevated circulating sPRR levels were inversely correlated with ankle–brachial index, an indicator of severe atherosclerosis of the lower limbs) in hemodialysis patients[220] ^65 and positively correlated with MMP2/MMP9 levels in patients with atherosclerosis.[221] ^21 These results imply the potential impact of sPRR on atherosclerosis progression and atherosclerotic plaque stability. In this way, elevated plasma sPRR levels may lead to vulnerable plaque. Similar to our previous report on the inhibitory effect of ELA‐32 on the PRR system,[222] ^31 here we found a reduced PRR protein level in CD68‐positive cells within aortic roots and plasma sPRR concentrations in HFD‐fed ApoE ^−/− mice with ELA‐21 treatment. Therefore, the antiatherosclerotic role of ELA‐21 may be related to the reduction of sPRR generation, which may further block AT1R‐mediated macrophage inflammation and foaming; this is awaiting further experimental clarification. We acknowledge that the present study has several shortcomings. On the one hand, the cohort study was a single‐center observational study with a relatively small case number and lacking a healthy control group. A significant age difference between the atherosclerosis and nonatherosclerosis groups was observed. Age might influence the plasma ELA levels. The correlation of ELA with the severity of atherosclerosis should be strengthened by adding other markers, such as the patients' ankle–brachial index. Future large‐scale and multicenter studies and longitudinal follow‐up studies are recommended to clarify the relationship between plasma ELA levels and patients with atherosclerosis' diagnosis, prognosis, mortality, and morbidity. On the other hand, the specific receptor mediating the antiatherosclerotic role of ELA‐21 and the impact of macrophage APJ on atherosclerosis is still unknown. The reasons for the simultaneous stimulation of ELA‐21 on the protein expression of macrophage ACE and ACE2 within plaques are unclear, because ELA has always been considered to inhibit ACE and PRR, and stimulate ACE2 in cardiovascular tissues. Future studies are also needed to determine the relative contribution of the elevated macrophage ACE and ACE2 to the antiatherosclerotic actions of ELA‐21. Nevertheless, our study, for the first time, showed the diagnostic and therapeutic potential of ELA in atherosclerosis. ELA may be an effective diagnostic biomarker and a novel candidate therapeutic target for treating atherosclerosis. Sources of Funding This work was supported by grants from the National Natural Science Foundation of China (numbers 82160051 and 32 100 908), the Jiangxi Provincial Natural Science Foundation (number 20232BAB206018), the Science and Technology Research Project in Education Department of Jiangxi Province (number GJJ2200904), Jiangxi Double Thousand Plan (number jxsq2020101074), the Ph.D. Start‐Up Research Fund in Jiangxi University of Chinese Medicine (number 2020BSZR009), the Science and Technology Research Project in Health Commission of Jiangxi Province (number 202311143), the Jiangxi Province Key Laboratory of Traditional Chinese Medicine for Cardiovascular Diseases (number 2024SSY06301), and the Scientific and Technological Innovation Team grant of the Jiangxi University of Chinese Medicine (number CXTD22014). Disclosures None. Supporting information Tables S1–S6. Figures S1–S5. [223]JAH3-14-e041261-s001.pdf^ (1.3MB, pdf) Acknowledgments