
==== Front
Medicine (Baltimore)
Medicine (Baltimore)
MD
Medicine
0025-7974
1536-5964
Lippincott Williams & Wilkins Hagerstown, MD

39029045
MD-D-24-00089
00047
10.1097/MD.0000000000038896
3
3400
Research Article
Clinical Trial/Experimental Study
Role of legumain in metabolic dysfunction, diagnosis, and prognosis of patients with atherosclerosis
Li Jingchao BD xiaoping@126.com
a
Li Kun BD xiaoping@126.com
a
Wang Huiqi BD lele121@163.com
a
Wang Le BD lele121@163.com
a
Li Xiaoping BD xiaoping@126.com
a
https://orcid.org/0009-0008-7855-267X
Liu Feifei BD a*
a The Second Department of Neurology, Circulation Medicine, Department of Infectious Diseases, Medical Records Room of Red Flag Hospital Affiliated to Mudanjiang Medical College, Mudanjiang, China.
* Correspondence: Feifei Liu, No. 5, Aimin District, Mudanjiang, Heilongjiang Province 157011, China (e-mail: feifeiliu0103@126.com).
19 7 2024
19 7 2024
103 29 e3889603 1 2024
19 6 2024
20 6 2024
Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal.

Background:

The purpose of this study was to investigate the role of legumain in metabolic dysfunction, diagnosis, and prognosis in patients with atherosclerosis.

Methods:

Plasma levels of legumain from patients with atherosclerosis (n = 320) and healthy controls (n = 320), expression of legumain in atheromatous plaque and secreted from monocyte-derived macrophages were measured using enzyme-linked-immunosorbent assay, reverse transcription-polymerase chain reaction, Western blot, immunohistochemistry, and fluorescence.

Results:

Data demonstrated that atherosclerotic patients had higher plasma level of legumain than healthy controls, which was a diagnostic and prognostic marker and corrected with the degree of atherosclerosis. It found that atheromatous plaque and endothelial cell had higher legumain expression than non-atherosclerotic arteries (controls). Legumain showed significantly increased secretion from pro-inflammatory M1 compared to pro-resolving M2 macrophages during monocyte-derived macrophages, which was localized to structures resembling foam cells.

Conclusion:

In conclusion, our data indicate that legumain expression is upregulated in both plasma and plaques of patients with atherosclerosis, which is associated with metabolic dysfunction of endothelial cell and might be a diagnostic and prognostic marker of atherosclerosis.

atherosclerosis
diagnosis
legumain
metabolic dysfunction
prognosis
OPEN-ACCESSTRUE
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pmc1. Introduction

Atherosclerosis is a chronic disease of the arterial wall that is a leading cause of death worldwide.[1] Clinically, atherosclerosis is characterized by many complex processes in vascular pathological lesion including vascular injury, inflammation, monocyte adhesion to endothelial cells, lipid deposition within macrophage foam cells, vascular oxidative stress injury, and neointimal hyperplasia.[2–4] Atherosclerosis is associated to oxidative stress and endothelial dysfunction.[5] Research into the atherosclerotic disease has resulted in many hypotheses about the pathophysiology and atherosclerosis-related complications such as heart failure, myocardial infarction, and stroke.[6] Therefore, understanding how to diagnose the degree of atherosclerosis will provide basic knowledge about atherosclerosis and can lead to new clinical applications for atherosclerotic diagnosis and prognosis.

Legumain, known as asparagine endopeptidase, is a lysosomal cysteine protease that belongs to peptidase family C13.[7] Legumain is related to cysteine cathepsins and plays an essential role during inflammation in human diseases.[8] Currently, a study has showed that legumain is increased in both plasma and carotid plaques in patients with carotid atherosclerosis.[9] Legumain increases the migration of human monocytes and human umbilical vein endothelial cells.[10] In addition, high expression of legumain is observed in atherosclerotic patients and may be a new and early biomarker of atherosclerosis.[11] Furthermore, legumain is presented in plaques, and its expression is upregulated in macrophages treated with oxidized low-density lipoprotein (oxLDL), which decreases oxLDL-induced macrophage apoptosis and the expression of inflammation. Therefore, it is crucial to investigate the diagnostic and prognostic role of legumain in patients with atherosclerosis.

In this study, we investigated the role of legumain in metabolic dysfunction, diagnosis, and prognosis in patients with atherosclerosis. The present study revealed that upregulation of legumain led to metabolic dysfunction in endothelial cell, which contributed to the formation of atheromatous plaque and secretion from pro-inflammatory M1 compared to pro-resolving M2 macrophages during monocyte-derived macrophages, suggesting that legumain may be a promising diagnostic and prognostic marker for atherosclerotic patients.

2. Materials and methods

2.1. Study design and patients

This clinical study was approved by the Ethical Committee of Red Flag Hospital Affiliated to Mudanjiang Medical College (approval number: RFHMDJ20180508-01). Recruitment for this study started in June 2018 and it was completed in July 2022. The study included 320 patients (54.8 ± 10.2 years of age) with atherosclerotic coronary artery disease and 320 age-matched 320 healthy individuals (55.3 ± 11.7 years of age). Patients were diagnosed aortic atherosclerosis (plaque thickness >2 mm) by multidetector computed tomography angiography. Inclusion criteria were as follows: (1) age between 40 and 85 years; (2) low-density lipoprotein cholesterol (LDL-C) ≥ 70 mg/dL or non-high-density lipoprotein cholesterol ≥100 mg/dL; (3) patients and single-vessel stenosis with moderate coronary lesions (10–90%). The key inclusion criteria for the trial were an age between 40 and 85 years, LDL-C ≥ 70 mg/dL or non-high-density lipoprotein cholesterol ≥100 mg/dL, and. Exclusion criteria were as follows: (1) autoimmune or diseases; (2) history of myocardial infarction, target vessel percutaneous coronary intervention, or coronary artery bypass graft; (3) severe hepatic or renal failure; (4) acute coronary syndrome. A follow-up was performed 360 days after study investigation for a standard virus panel and to assess prognosis. The subjects visited the study clinic every 30 days. The inclusion criteria of the healthy individuals were as follow: (1) age between 40 and 85 years; (2) participants with normal lipid index; (3) participants without cancers; (4) participants without cardiac surgery; (5) participants without mental disease. All the subjects signed written informed consent.

2.2. Enzyme-linked-immunosorbent assay

A total of 5 mL blood samples were collected from participants. Plasma level of legumain in samples was measured by enzyme-linked-immunosorbent assay (Cat. no DY4749, R&D Systems, Minneapolis, MN) according to the manufacturers guidance.

2.3. Analysis of endothelial function and cell viability

Endothelial cell were treated with legumain (1 mM, Sigma-Aldrich), legumain inhibitor (LI-1, 1 mM, Sigma-Aldrich) or PBS (Control) for 12 hours at 37 °C. Endothelial cells function was determined as described previously.[12] The viability of endothelial cells was analyzed using the Cell Counting KIT-8 (#CK04-100 T; Solarbio) according to the manufacturer’s protocol.

2.4. Legumain activity

The activity of legumain in atheromatous plaque and endothelial cells was measured using an Amylase Activity Assay Kit (Sigma, Sydney, Australia) according to the manufacturer’s protocol.

2.5. Statistical analysis

Data are expressed as means ± standard deviations (SD) or n (%). All statistical analyses were performed using SPSS version 19.0 (Chicago, IL). Significant differences were analyzed using Student t test between 2 groups and analysis of variance (ANOVA) followed by Tukey post hoc test for multiple comparisons between multiple groups. Correlation analyses were performed by Spearman test. Significance was at P < .05.

3. Results

3.1. Characteristic of patients with atherosclerosis

A total of 320 patients with atherosclerosis and age-matched 320 healthy controls were enrolled in this study. The number of male atherosclerotic patients (n = 188) was more than female atherosclerotic patients (n = 132). The baseline and clinical characteristics of the atherosclerotic patients are shown in Table 1. Patients with atherosclerosis received optimal medical therapy such as b-blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers (ACE-I/ARB), statin medicines, antiplatelet drugs, and organic nitrate drugs. There was no significant difference in terms of age, sex, and body mass index (BMI) between atherosclerotic patients and healthy individuals.

Table 1 Baseline characteristic of patients with atherosclerosis.

Parameter	Atherosclerotic patients	Healthy control	P value	
Number	320	320	‐	
Sex				
Male	188 (58.8)	180 (56.3)	.085	
Female	132 (41.2)	140 (43.7)	.088	
Age (years)	54.8 ± 10.2	55.3 ± 11.7	.076	
Body mass index (kg/cm2)	32.2 ± 2.6	22.6 ± 2.0	.008	
Blood pressure systolic (mm Hg)	136 ± 14	118 ± 10	.006	
Blood pressure diastolic (mm Hg)	84 ± 10	80 ± 8	.032	
Low-density lipoprotein (mg/dL)	82.0 ± 30.0	30.0 ± 15.0**	.001	
Triglycerides (mg/ dL)	143.0 ± 26.0	43.0 ± 20.0**	.001	
Fasting blood glucose (mg/dL)	138.0 ± 36.0	106.0 ± 22.0**	.003	
Creatinine (mg/ dL)	1.2 ± 0.4	0.8 ± 0.2*	.040	
Aspartate-aminotransferase (U/L)	33.5 ± 14.0	17.8 ± 7.2**	.003	
Alanine-aminotransferase (U/L)	40.6 ± 26	20.4 ± 8.6**	.006	
HbA1c (mmol/mol)	50.6 ± 24.0	24.5 ± 12.7*	.026	
β-Blocker	60 (18.7)	0	‐	
ACE-I/ARB	53 (16.6)	0	‐	
Antiplatelet drug	40 (12.5)	0	‐	
Statin	88 (27.5)	0	‐	
Organic nitrate drugs	79 (24.7)	0	-	
Data are expressed mean ± SD or n (%).

* P < .05.

** P < .01 vs healthy control.

3.2. Analysis of plasma level of legumain in atherosclerotic patients

The diagnostic role of legumain was investigated in atherosclerotic patients. As shown in Table 2, the mean level of plasma legumain was significant higher in patients with atherosclerosis than healthy individuals (**P < .01). We revealed that mRNA level of legumain was upregulated in PBMCs in atherosclerotic patients compared to healthy control. There were no significant differences between male and female atherosclerotic patients (Fig. 1A). Data demonstrated that plasma level of legumain was positively associated with the degree of atherosclerosis (Fig. 1B). These data suggest that legumain has higher plasma level and a diagnostic marker for patients with atherosclerosis.

Table 2 Plasma level of legumain in atherosclerotic patients.

	Atherosclerotic patients	Healthy control	P value	
Legumain level (ng/mL)	3.52 ± 1.14	1.56 ± 0.36**	.002	
mRNA level (Fold)	2.36 ± 0.30	1.0 ± 0.16**	.004	
Data are expressed mean ± SD.

** P < .01 vs. healthy control.

Figure 1. Measurement of plasma level of legumain in atherosclerotic patients. (A) Plasma level of legumain in atherosclerotic patients and healthy individuals. (B) Association between plasma level of legumain and degree of atherosclerosis. **P < .01 versus healthy individuals.

3.3. Expression of legumain in atheromatous plaque and endothelial cell

The expression of legumain was analyzed in atheromatous plaque and endothelial cell in this study. Outcomes showed that legumain was abundantly expressed in atheromatous plaques in atheromatous plaques in patients with atherosclerosis compared to healthy control (Fig. 2A). Data also showed that legumain was higher expressed in endothelial cell than non-atherosclerotic arteries (Fig. 2B). Legumain activity was also upregulated in atheromatous plaque (Fig. 2C). Also, legumain activity was higher in endothelial cells in patients with atherosclerosis compared to healthy control (Fig. 2D). These data indicate that legumain is overexpressed in atheromatous plaque and endothelial cell in patients with atherosclerosis.

Figure 2. Expression level of legumain in atheromatous plaque and endothelial cell. (A) Legumain expression in atheromatous plaques in patients with atherosclerosis determined by immunohistochemistry. (B) Legumain expression in endothelial cell in patients with atherosclerosis determined by immunofluorescence. (C and D) Relative legumain activity in atheromatous plaque (C) and endothelial cells (D). **P < .01.

3.4. Effect of legumain on metabolic function in endothelial cell

We analyzed the legumain on metabolic function in endothelial cell in vitro. As shown in Figure 3A, legumain decreased endothelial cell viability. There were significant differences were observed in the change of endothelial function parameters including Global Arginine Bioavailability Ratio (GABR), arginine-to-ornithine-ratio (AOR), asymmetric dimethylarginine (ADMA), serum soluble intercellular adhesion molecule-1 (sICAM-1) and serum soluble vascular cell adhesion molecule-1 (sVCAM-1) between legumain and control group (Figs. 3B–D). Legumain inhibitor (LegumainIR, LI-1 inhibitor) reversed the effect of legumain on metabolic function of endothelial cells. These data suggest that legumain decreases metabolic function of endothelial cells.

Figure 3. Effect of legumain on metabolic function in endothelial cell. (A) Viability of endothelial cell after treatment with legumain or legumain inhibitor. (B) Effect of legumain or legumain inhibitor on Global Arginine Bioavailability Ratio (GABR) (P = .608), arginine-to-ornithine-ratio (AOR, P = .549) in endothelial cells. (C) Effect of legumain or legumain inhibitor on asymmetric dimethylarginine (ADMA) in endothelial cells. (D) Effect of legumain or legumain inhibitor on serum soluble intercellular adhesion molecule-1 (sICAM-1) and serum soluble vascular cell adhesion molecule-1 (sVCAM-1) in endothelial cells. **P < .01.

3.5. Expression of legumain in monocyte-derived macrophages

The expression of legumain in monocyte-derived macrophages was investigated in this study. As shown in Figure 4A, legumain showed significantly increased secretion from pro-inflammatory M1 compared to pro-resolving M2 macrophages during monocyte-derived macrophages. Data also showed that legumain was localized to structures resembling foam cells (Fig. 4B). Compared to macrophages, legumain expression was relatively high in pro-inflammatory M1 and M2. These results indicate that risk stratification of the legumain level could guide treatment selection in monocyte-derived macrophages for patients with atherosclerosis.

Figure 4. Legumain is expressed in foam-like cells in carotid plaques and localized to both M1 and M2 macrophages within carotid plaques. (A) Legumain expression in M1 and M2 macrophages within carotid plaques. (B) Legumain expression localized to structures resembling foam cells. **P < .01.

3.6. Association between legumain and prognosis in patients with atherosclerosis

We further analyzed the association between legumain expression and prognosis in patients with atherosclerosis. As shown in Figure 5, serum level of legumain was negatively corrected with the prognosis of patients with atherosclerosis. These data suggest that legumain is a prognostic marker of atherosclerosis.

Figure 5. Association between plasma level of legumain and prognosis in patients with atherosclerosis.

4. Discussion

In this study, we investigated the role of legumain on the diagnosis, prognosis and metabolic dysfunction in patients with atherosclerosis. Data in this study found that legumain was upregulated in plasma in patients with atherosclerosis compared to healthy individuals, which was a potential diagnostic marker for atherosclerotic patients. In addition, legumain improved the endothelial function parameters including GABR, AOR, ADMA, sICAM-1, and sVCAM-1 in arterial endothelial cell, which are key events in the progression of atherosclerosis. Furthermore, data showed that legumain is positively corrected with degree of atherosclerosis and negatively corrected with prognosis of atherosclerotic patients.

Evidence has indicated that legumain is increased in both plasma and plaques of patients with carotid stenosis and might be a new and early biomarker of atherosclerosis.[11] Legumain regulates oxLDL-induced macrophage apoptosis by enhancing the autophagy pathway, which may also influence the vulnerability of atherosclerotic plaques. Data in this study demonstrated that legumain is overexpressed in atherosclerotic plaques and endothelial cells compared to non-atherosclerotic arteries. Even though we did observe the correction between plasma level of legumain and degree of atherosclerosis, we did not observe in this population study a positive correlation between legumain expression and endothelial function. We focused on effect of legumain on endothelial function in vitro, while we did not observe linear relation of vascular function and legumain expression.

Oxidation of LDL-C is one of the key factors for the development of atherosclerosis, which has a high affinity for macrophages in patients with atherosclerosis.[5,13,14] Activated endothelial cells from damaged endothelium express cytokines, chemokines, and adhesion molecules attract circulating monocytes toward the atherosclerotic lesion that further induce the maturation of monocytes into M1 phenotype pro-inflammatory macrophages.[15] In addition, accumulation of oxLDL leads to formation of foam cells within the macrophages located in the intima in response to activated endothelial cells by inflammation.[16–18] Furthermore, previous study suggests that the active legumain produced by M2 macrophages is found to be overexpressed in cellular expression and processing.[10] In this study, we observed that risk stratification of the legumain level could guide treatment selection in monocyte-derived macrophages for patients with atherosclerosis. Interestingly, we indicate that legumain is a prognostic marker of atherosclerosis.

Several limitations of the study should address. First, this study did not investigate the correlation between legumain and age of atherosclerotic patients. Second, correlations between expression of legumain and the severity of atherosclerosis were not analyzed in this study. Third, the effect of legumain on lipid metabolism and atherosclerosis risk did not investigate in atherosclerotic patients. Therefore, further study should be conducted to investigate the association between the severity of atherosclerosis, patients age, atherosclerotic treatment outcomes, and expression of legumain.

5. Conclusion

In conclusion, this study demonstrates a diagnostic and prognostic effect of legumain via measurements of plasma concentration in patients with atherosclerotic disease. Data indicate that legumain is associated with metabolic function of endothelial cell via various measurements of endothelial function. Whether legumain can be regarded as a novel therapeutic target for the cardiovascular disease, needs further investigation.

Author contributions

Conceptualization: Jingchao Li, Huiqi Wang, Feifei Liu.

Data curation: Jingchao Li, Xiaoping Li, Feifei Liu.

Formal analysis: Jingchao Li, Huiqi Wang, Le Wang.

Investigation: Huiqi Wang.

Methodology: Jingchao Li, Huiqi Wang, Xiaoping Li.

Project administration: Jingchao Li, Le Wang, Xiaoping Li, Feifei Liu.

Resources: Kun Li, Huiqi Wang, Le Wang, Xiaoping Li, Feifei Liu.

Software: Kun Li, Huiqi Wang, Le Wang, Feifei Liu.

Supervision: Kun Li.

Validation: Kun Li.

Visualization: Kun Li, Xiaoping Li, Feifei Liu.

Writing – original draft: Kun Li, Feifei Liu.

Writing – review & editing: Feifei Liu.

Abbreviations:

LDL-C low-density lipoprotein cholesterol

oxLDL oxidized low-density lipoprotein

The authors have no conflicts of interest to disclose.

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

How to cite this article: Li J, Li K, Wang H, Wang L, Li X, Liu F. Role of legumain in metabolic dysfunction, diagnosis, and prognosis of patients with atherosclerosis. Medicine 2024;103:29(e38896).
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References

[1] Lim DH Lee Y Park GM . Serum uric acid level and subclinical coronary atherosclerosis in asymptomatic individuals: an observational cohort study. Atherosclerosis. 2019;288 :112–7.31352272
[2] Ma YH Leng XY Dong Y . Risk factors for intracranial atherosclerosis: a systematic review and meta-analysis. Atherosclerosis. 2019;281 :71–7.30658194
[3] Gupta KK Ali S Sanghera RS . Pharmacological options in atherosclerosis: a review of the existing evidence. Cardiol Ther. 2019;8 :5–20.30543029
[4] Wang P Xu YY Lv TT . Subclinical atherosclerosis in patients with type 1 diabetes mellitus: a systematic review and meta-analysis. Angiology. 2019;70 :141–59.30009613
[5] Marchio P Guerra-Ojeda S Vila JM Aldasoro M Victor VM Mauricio MD . Targeting early atherosclerosis: a focus on oxidative stress and inflammation. Oxid Med Cell Longevity. 2019;2019 :8563845.
[6] Fava C Montagnana M . Atherosclerosis is an inflammatory disease which lacks a common anti-inflammatory therapy: how human genetics can help to this issue. Front Pharmacol. 2018;9 :55.29467655
[7] Zhen Y Chunlei G Wenzhi S . Clinicopathologic significance of legumain overexpression in cancer: a systematic review and meta-analysis. Sci Rep. 2015;5 :16599.26607955
[8] Dall E Brandstetter H . Structure and function of legumain in health and disease. Biochimie. 2016;122 :126–50.26403494
[9] Bai P Lyu L Yu T . Macrophage-derived legumain promotes pulmonary hypertension by activating the MMP (Matrix Metalloproteinase)-2/TGF (Transforming Growth Factor)-beta1 signaling. Arterioscler Thromb Vasc Biol. 2019;39 :e130–45.30676070
[10] Solberg R Smith R Almlof M Tewolde E Nilsen H Johansen HT . Legumain expression, activity and secretion are increased during monocyte-to-macrophage differentiation and inhibited by atorvastatin. Biol Chem. 2015;396 :71–80.25205715
[11] Lunde NN Holm S Dahl TB . Increased levels of legumain in plasma and plaques from patients with carotid atherosclerosis. Atherosclerosis. 2017;257 :216–23.27940038
[12] Tripolt NJ Aberer F Riedl R . Effects of linagliptin on endothelial function and postprandial lipids in coronary artery disease patients with early diabetes: a randomized, placebo-controlled, double-blind trial. Cardiovasc Diabetol. 2018;17 :71.29773079
[13] Liu M Yang W Liu S . LXRalpha is expressed at higher levels in healthy people compared to atherosclerosis patients and its over-expression polarizes macrophages towards an anti-inflammatory MPhi2 phenotype. Clin Exp Hypertens. 2018;40 :213–7.29420090
[14] Zhang T Shao B Liu GA . Rosuvastatin promotes the differentiation of peripheral blood monocytes into M2 macrophages in patients with atherosclerosis by activating PPAR-gamma. Eur Rev Med Pharmacol Sci. 2017;21 :4464–71.29077145
[15] Medina-Leyte DJ Zepeda-Garcia O Dominguez-Perez M Gonzalez-Garrido A Villarreal-Molina T Jacobo-Albavera L . Endothelial dysfunction, inflammation and coronary artery disease: potential biomarkers and promising therapeutical approaches. Int J Mol Sci . 2021;22 :3850.33917744
[16] Yan D He Y Dai J Yang L Wang X Ruan Q . Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation. Biosci Rep. 2017;37 :01.
[17] Suzuki Y Tada-Oikawa S Ichihara G . Zinc oxide nanoparticles induce migration and adhesion of monocytes to endothelial cells and accelerate foam cell formation. Toxicol Appl Pharmacol. 2014;278 :16–25.24746987
[18] Liu HJ Wang XL Zhang L . Inhibitions of vascular endothelial growth factor expression and foam cell formation by EGb 761, a special extract of Ginkgo biloba, in oxidatively modified low-density lipoprotein-induced human THP-1 monocytes cells. Phytomedicine. 2009;16 :138–45.19135347
