https://orcid.org/0000-0002-3573-6638
Abstract
Atherosclerosis is a lipid-driven chronic inflammatory disease that poses a serious threat to health. Legumain (LGMN), also known as asparagine endonuclease, is a new type of cysteine proteases that can specifically hydrolyze substrate molecules containing asparagine residues. It has anti-apoptotic effects in mammals and plays an antigen-presenting role in inflammatory response. Several studies have found that LGMN can activate multiple signal pathways to promote cell apoptosis and migration, inflammatory response, and the development of atherosclerosis. Importantly, LGMN exerts pro-atherogenic effects by participating in a variety of pathophysiological mechanisms of atherosclerosis, including vascular remodeling, inflammatory response, plaque stability, and the degradation of extracellular matrix. In the present review, we describe the LGMN distribution, structure, generation, and functional partners. Furthermore, we summarize the relationship between LGMN and atherosclerosis. Based on the relationship between LGMN and atherosclerosis, LGMN may be a potential biomarker for atherosclerosis.
Introduction
Atherosclerosis is a major cause of coronary artery disease, stroke, and peripheral arterial disease, which poses a serious threat to human health.[1] Epidemiological studies indicated that the prevalence of atherosclerosis is significantly increased with the adoption of western lifestyles.[2, 3] To solve this thorny problem, researchers have made various attempts and efforts, however, the expected results have not been obtained so far.[4] It is well-accepted that atherosclerosis is a lipid-driven chronic inflammatory disease in the vascular wall. Atherosclerosis pathogenesis is very complex, and it mainly includes endothelial cell injury, lipid infiltration, and inflammatory mediator secretion, which eventually leads to the formation of a large number of plaques in large- and middle-sized arteries and eventually leads to cardiovascular disease.[5–7] To date, there are various therapeutic strategies for organ fibrosis, such as atorvastatin, simvastatin, and so on. Although statins are widely used in clinical practice to treat atherosclerosis, the cardiovascular risk remains a serious challenge due to their limitations.[8] Consequently, a detailed understanding of the key molecules involved in atherogenesis is essential for us to further understand its pathogenesis and search for novel therapeutic targets.
Legumain (LGMN), also known as asparagine endopeptidase (APE) to emphasize its specific function, is a “new” member of the Clusters of Differentiation (CD) family of cysteine proteases present in lysosomes with a high specificity for hydrolyzing substrate molecules containing asparagine residue sites, which is the specificity of LGMN.[9, 10] Early in the 1980s, it was identified in plants as a processing enzyme for storage proteins during seed germination,[11] and subsequently also in parasites[12] and mammals.[13] In mammalian genome, LGMN is the only cysteine protease that specifically cleaves the peptide bond of asparagine residues.[14] Although several isoforms of LGMN are found in plants, only one is found in mammals (including humans), suggesting that LGMN may play a more important role in mammals.[15] LGMN is ubiquitously expressed in mammalian tissues, such as the liver, kidney, lung, and brain.[15, 16] In addition, LGMN can be localized to different types of antigen-presenting cells, including human and mouse dendritic cells and macrophages, etc. The expression of LGMN in mouse B-cell lines was also abundant, suggesting the immunological significance of LGMN.[17, 18] LGMN is involved in a variety of pathophysiological processes in the mammalian organisms, including osteolysis, inflammatory response, tumorigenesis, and atherosclerosis.[19]
Studies have shown that LGMN is involved in many pathophysiological processes, such as atherosclerosis,[20, 21] pulmonary hypertension,[22] and liver fibrosis.[23] Immune-mediated inflammatory response is one of the main mechanisms of atherosclerosis. LGMN is involved in the processing and presentation of antigens in the immune process. LGMN is associated with the formation of atherosclerosis and plaque stability. It plays an important role in the process of atherosclerosis. Notably, it has been shown that LGMN is involved in the development of atherosclerosis through multiple mechanisms. Moreover, evidence from several perspectives show that LGMN aggravates atherosclerosis by atherosclerotic vascular remodeling (VR), degradation of extracellular matrix (ECM), and plaque vulnerability.[20, 24] Papaspyridonos et al. used a whole-transcriptome approach to identify LGMN in 18 genes associated with plaque rupture, which may be a new target for the treatment of atherosclerotic plaques or a diagnostic marker for plaque development.[9] The demands for prevention and therapeutic strategy are in conjunction with these pathological behaviors of LGMN on the development of atherosclerosis. Therefore, we provide a comprehensive description of LGMN distribution, structure, generation, and functional partners. Furthermore, we summarize the relationship between LGMN and atherosclerosis, and then rationally asses its application value in cardiovascular diseases to provide an alternative idea for future diagnosis and treatment.
A brief overview of legumain
The distribution of legumain
According to sequence similarity, LGMN is a highly conserved protein among different species, including Homo sapiens (human), Pongo abelii (ponb), Bos taurus (bovin), Mus musculus (mouse), and Rattus norvegicus (rat). According to the Human Protein Atlas (HPA) database (http://www.proteinatlas.org), LGMN is widely expressed in human tissues (Fig. 1A).
The distribution of LGMN. (A) Comparison of LGMN amino acid sequences among Homo sapiens (human), Pongo abelii (ponb), Bos taurus (bovin), Mus musculus (mouse), and Rattus norvegicus (rat). The degree of conservation of LGMN amino acid sequences among different species was analyzed by ESPript. Conservative residues are circled. Residues whose expression is constant in different species have been marked. The similarity of the amino acid sequence of LGMN is 93.84% among different species. (B) The subcellular localization of LGMN evaluated by COMPARTMENT database. (C) The tissue distribution of the LGMN mRNA from HPA.
In the subcellular, LGMN can be found in the lysosome, extracellular, endosome, cytosol, endoplasmic reticulum (ER), nucleus, cytoskeleton, and plasma membrane, which is obtained from COMPARTMENTS databases (https://compartments.jensenlab.org/) (Fig. 1B). Based on the HPA database (http://www.proteinatlas.org/), LGMN mRNA is mainly distributed in the brain, endocrine tissues, proximal digestive tract, kidney, lung, endocrine tissues, liver, adipose tissue, pancreas, and gastrointestinal tract (Fig. 1C). All these analyses imply that LGMN is highly conserved and widely distributed in human tissues.
The functional partners with legumain
In the STRING database (https://cn.string-db.org/), the predicted functional partners with LGMN include cystatin-M (CST6), Human leukocyte antigen (HLA) class II histocompatibility antigen gamma chain (CD74), major histocompatibility complex, class II, DQ alpha 1 (HLA-DQA1), toll-like receptor 7, cubilin (CUBN), HLA class II histocompatibility antigen, DRB1-15 beta chain (HLA-DRB1), major histocompatibility complex, class II, dr alpha (HLA-DRA), major histocompatibility complex, class II, dp alpha 1 (HLA-DPA1), major histocompatibility complex, class II, dq beta 1 (HLA-DQB1), major histocompatibility complex, class II, DQ beta 2 (HLA-DQB2) (Fig. 2).
The PPI information about LGMN for predicted functional partners was evaluated by STRING database.
The structure and generation of legumain
The LGMN gene was highly conserved in different species, and the gene encoding human LGMN is located on chromosome 14 at 14q32.12, consisting of 14 exons and 13 introns, encoding a polypeptide chain composed of 433 amino acid residues, which after glycation becomes a mature enzyme with biological activity.[10] LGMN has a highly conserved His148-Gly-spacer-Ala-Cys189 gene sequence, while other proteins in the CD family also have the same motif, so it was classified as the CD family.[10] The structure of the pro-LGMN is composed of three parts: the signal peptide (SP), the catalytic function region, and the stabilization and activation regulation region (LSAM) (Fig. 3).[25] Among them, SP guides LGMN to the ER for processing.[26] The catalytic functional region stabilizes the enzyme activity of LGMN by three spatially close active site residues, His148, Cys189, and Asn42, which are also critical to perform a hydrolytic function. LSAM ensures the stable state of the LGMN precursor by triggering an electrostatic-coded stabilization switch located near the catalytic domain.[26] The C-terminal pro-domain can be subdivided into activating peptide (AP) and LSAM. AP is a latency-conferring activation peptide that blocks access to non-primers, and LSAM is the primer binding site. These structures are the reason why pro-LGMN is inactive.[26, 27] Chen et al. showed by co-immunoprecipitation that there were three forms of human LGMN protein, the 56 kDa pro-LGMN, the mature enzyme of 46 kDa and 36 kDa, among which the pro-LGMN was inactive.[28] In mammals, LGMN is synthesized as an inactive pro-LGMN, where a change in acidic pH triggers autocatalytic mechanisms to convert to an active form of the 46 kDa mature enzyme.[25] At pH 5.5, processing of C-terminal Asn323 was observed in mammals, while processing of N-terminal Asp21 and Asp25 occurred only at pH ≤ 4.5. N-terminal cleavage is not required for activation, although post-treatment of Asn323 residues is critical for producing peptidase activity.[29] The mature enzyme of 46 kDa is converted to 36 kDa when processed by various proteases in the lysosome. Interestingly, the enzymatic activities of the 36 kDa and 46 kDa peptide substrates were virtually identical.[25]
The structure and generation of LGMN. SP: signal peptide, NTF: N-terminal structural domain, AP: activating peptide, LSAM: stabilization and activation regulatory region, and AEP: endopeptidase.
Basic biological characteristics of legumain
In mammals, LGMN also plays an important role in the immune system. In particular, LGMN is involved in the processing of self and foreign proteins, in major histocompatibility complex II and antigen presentation, so that it is involved both in the development of tolerance through the destruction of self-proteins and in the increased immune response to foreign antigens through their processing.[30] The main mechanism of action of LGMN is to participate in the process of antigen presentation. Ju et al. used recombinant LGMN to express in Escherichia coli, and confirmed that recombinant LGMN had the function of resisting serum antigens of patients with liver fluke.[31] Manoury et al. added tetanus toxin antigen to phagocytic lysosomes extracted from human B cell lines and found that LGMN plays a major role in antigen processing.[32, 33] At the same time, the study also found that in B cells, LGMN can initiate the dissociation of the constant chain, and inhibition of LGMN activity by inhibitors inhibits the processing of antigenic peptides that replace the constant chain and bind to the grooving region of the major histocompatibility complex molecule, while the dissociation process of the constant chain is also significantly slowed down. These studies have shown that LGMN is involved in antigen processing and presentation during the immune process. In addition to playing an immune role in antigen processing, LGMN is also an important factor in immune signal transduction. It can activate toll-like receptors by enzyme digestion like cathepsin and participates in innate immunity.[34]
Role of legumain in atherosclerosis
Atherosclerosis is a complex process induced by multiple factors. LGMN has been shown to affect many processes of atherogenesis, such as VR, inflammatory response, ECM degradation, and macrophage apoptosis (Fig. 4), as discussed below.
The pro-atherogenic action of LGMN. As: atherosclerosis, MCP-1: monocyte chemoattractant protein-1, IL-6: interleukin-6, and ECM: extracellular matrix.
Legumain enhances atherosclerotic vascular remodeling
VR refers to chronic changes in the diameter of blood vessels or changes in the structure of blood vessel walls, which can be divided into positive remodeling and negative remodeling.[35] The occurrence of atherosclerosis is accompanied by abnormal VR due to the disturbance of lipid metabolism and the release of various inflammatory factors in the inflammatory response. It not only affects the degradation of ECM, such as collagen, elastin, fibronectin, and matrix metalloproteinases (MMPs), but also further acts on vascular wall cells, promotes the proliferation, migration and apoptosis of endothelial cells, smooth muscle cells and fibroblasts, and re-forms the intima.[36] Ozawa et al. observed that LGMN boosted Vascular Smooth Muscle Cell (VSMC) migration, whereas LGMN (10, 50 ng/ml) significantly improved ox-low-density lipoprotein (LDL)-induced macrophage foam cell production. In vitro, LGMN was found to increase collagen-3, fibronectin, and elastin expression in VSMC. In vivo, during the formation of atherosclerotic lesions, it was found that LGMN increased the expression of collagen-3 in VSMC in ApoE−/− mice, thereby promoting atherosclerotic VR.[24] Whether promoting the formation of macrophage foam cells or increasing the production of collagen-3, fibronectin, and elastin by VSMCs, all of which contribute to the development of atherosclerosis. What’s more, studies also found that LGMN promoted the expression of ECM proteins, such as phosphoinositide 3-kinase (PI3K), Akt phosphorylation, and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. These indicated that LGMN promotes atherosclerotic VR as a result of increased ECM production via the PI3K/Akt pathway.[24]
Infiltration of inflammatory factors, cell migration, and degradation of ECM has been shown to contribute to atherosclerosis.[37] Clerin et al. demonstrated that LGMN had the ability to induce monocyte migration by checkerboard analysis.[38] However, LGMN may regulate TLR4, mitogen-activated protein kinase, Drosophila antibiological skin growth factor proteinase 3 (Smad3), protein kinase B (Akt), and other signaling pathways to induce the maturation and migration of mononuclear macrophages, enhance inflammatory response, and promote the occurrence and development of atherosclerosis.[39, 40] Clerin et al. found that LGMN is expressed not only in macrophages but also in endothelial cells. Furthermore, the migration of Human umbilical vein endothelial cells (HUVECs) was induced by pro-LGMN detected by two different assays. In the wound healing assay, compared with HUVECs exposed to the control medium, the number of cells treated with LGMN on the initial wound area was significantly increased. Moreover, in a modified Boyden chamber assay, the number of HUVECs that invading a Matrigel-coated membrane increased in a dose-dependent manner, which was observed in response to LGMN.[38]
Legumain promotes inflammatory response
Atherosclerosis is a chronic inflammatory disease. Inflammation is not only the basis of various physiological and pathological processes but also the main cause of atherosclerosis, which runs through the occurrence and development of atherosclerosis.[41] Lunde et al. found that LGMN increases the expression of proto-typical pro-inflammatory factor monocyte chemoattractant protein-1 (MCP-1) mRNA, which is a key inflammatory factor and has a specific chemotactic activation effect on mononuclear macrophages and promotes inflammatory response.[24, 42] Further studies showed that LGMN also promoted the expression of Interleukin (IL)-6 mRNA, which is an important inflammatory factor involved in cardiovascular pathological processes and has a strong regulatory effect on ECM.[43, 44] Notably, pro-inflammatory cytokines such as IL-1β, Interferon (IFN)-γ, and Tumor Necrosis Factor (TNF)-α, which have been shown to increase the number of cysteine proteases in monocyte and vascular cells, also regulate LGMN gene expression.[45–47]
Legumain destabilizes atherosclerotic plaques
Atherosclerosis is a complex vascular wall lesion involving large and middle arteries. Its pathological features include lipid accumulation, inflammatory cell infiltration, smooth muscle cell migration into the intima and proliferation, and ultimately leading to plaque formation. Atherosclerotic plaque progression or plaque rupture is one of the main causes of cardiovascular events.[48, 49] LGMN is a novel cysteine protease, which can affect the remodeling of ECM and thus affect the progression of atherosclerotic lesions and the stability of plaque. LGMN activates other proteases, such as matrix metalloproteinase 2, cathepsin L, and fibronectin.[50–52] Several studies have reported up-regulated expression of LGMN mRNA and protein in regions of carotid atherosclerotic plaque instability, suggesting a role for LGMN in unstable plaques. Clerin et al. by microarray analysis found that the expression of mRNA that regulates LGMN increases with the progression of atherosclerosis in the aorta of aging ApoE−/− mice, while in the aorta of C57BL/6J mice of age-matched, the LGMN mRNA remains low and unchanged. LGMN was determined to be predominantly expressed in macrophages in atherosclerotic carotid, in lesions at the aortic sinus, in wounded carotid arteries of ApoE−/− mice, and in inflammatory areas in advanced human coronary atherosclerotic plaques, according to situ hybridization and immunohistochemistry research.[38] Fang et al. analyzed the rat thoracic aorta by immunofluorescence and immunohistochemistry and found that the expression of LGMN in plaques was significantly increased in the atherosclerotic group compared to the statin-treated group, and the expression of LGMN was mainly associated with mononuclear macrophages and foam cells. The results of western-blot analysis and reverse transcriptase coupled polymerase chain reaction results showed that the expression of LGMN protein and mRNA was significantly decreased after statin treatment.[53] In addition, in a Norwegian study, plasma LGMN levels were significantly higher in patients with carotid stenosis than in healthy controls, but there was no correlation between the degree of carotid artery stenosis and LGMN levels.[20] LGMN can degrade ECM, regulate leukocyte migration, and induce apoptosis of macrophages during the differentiation of monocytes into macrophages. While the degradation of ECM is carried out by proteases such as MMP and serine, which promote the rupture of atherosclerotic plaque, thereby causing plaque instability.[25, 54]
Legumain stimulates extracellular matrix degradation
ECM, including collagen fibers, proteoglycans, and elastin, is synthesized by smooth muscle cells in the plaque. The changes in ECM are closely related to the pathogenesis of atherosclerosis. The decrease of ECM synthesis and the increase of ECM degradation are the main intrinsic causes of plaque rupture.[55] However, MMPs play an essential role in both normal and pathological ECM remodeling.[56] Recent research has shown that LGMN regulates the remodeling of the ECM by inducing the degradation of collagen-1 and fibronectin in a variety of pathological situations.[52, 57–59] Mattock et al. used a novel Enzyme linked immunosorbent assay (ELISA) and modified activity assay to detect carotid plaque extracts from 17 patients and found that, compared with the stable region, the amount and activity of LGMN protein were more than twice in the unstable region of the plaque.[60] Shen et al. used the western-blot method to confirm that fibronectin and collagen-1 expression were significantly reduced after treatment with the LGMN inhibitor RR-11a.[59] LGMN promotes the degradation of ECM by activating the matrix metalloproteinase precursor 2 (pro-MMP-2) and cathepsin B, H, and L, or by directly increasing the hydrolysis of proteins (such as fibronectin) in ECM.[39, 52, 60] In conclusion, the activity of LGMN is significantly increased in the unstable areas of carotid atherosclerotic plaques, which may be one of the reasons for the plaque instability enhancement caused by LGMN.
Legumain and macrophage apoptosis
In the process of atherosclerosis, macrophages play a role by regulating lipid content, inflammatory response, degradation of fiber components, and neovascularization.[61] In advanced atherosclerosis, plaque necrosis is due to apoptosis of macrophages and defective clearance of apoptotic cells by phagocytes, further triggering acute atherosclerotic thrombotic cardiovascular events.[62] Cathepsin L, a cysteine protease with potent collagenolytic and elastinolytic activities, which is overexpressed in plaque. Moreover, it is closely associated with LGMN. Li et al. have demonstrated that the release of cathepsin into the cytoplasm can trigger macrophage apoptosis.[63, 64] In the early stages of atherosclerosis, expression of cathepsin L is localized to apoptotic macrophages, but is not found in other cells.[65] Cathepsin L is present in the fibrous cap, middle membrane, and macrophage-rich regions of atherosclerotic plaques.[45] During plaque stabilization, cathepsin L is associated with macrophage apoptosis and a mediator of matrix remodeling because it is highly expressed in plaques, and it is also the most prominent of enzyme processed by LGMN.[46, 64] Cathepsin L has the function of cleaving mature insoluble elastin.[66] In a mouse model of atherosclerosis, cathepsin L deficiency reduces the incidence of atherosclerosis.[67] In addition, Mattock et al. confirmed that cathepsin L was the most significantly expressed enzyme after being activated by LGMN. Cathepsin L normally has a relative molecular mass of 30 kDa, while it is only active when it is in the relative molecular mass of 25 kDa form.[60] Interestingly, LGMN activity affects the processing of cathepsin L in human macrophages. It was observed that cathepsin L existed only in single-stranded form (30 kDa), while not in double-stranded form (25 kDa) in LGMN knockout mice. Further studies revealed that LGMN was able to cleave the inactive single-stranded form of cathepsin L into the mature double-stranded form, which is highly expressed in atherosclerotic plaques and promotes apoptosis of macrophages.[51] Another study indicated that plaques size, macrophage and lymphocyte infiltration, levels of collagen are reduced, and medial elastin is degraded, which phenomena can be observed in cathepsin L and low-density lipoprotein receptor double-knockout mice.[67] What’s more, A study demonstrated that LGMN is closely related to autophagy. When LGMN was overexpressed, it not only reduced the expression of caspase-3, caspase-9 and Bax, but also promoted the expression of microtubule-associated proteins light chain 3 and yeast autophagy-related gene homolog of Atg6 (Beclin1). LGMN suppresses ox-LDL-induced apoptosis in macrophages, which in turn increases macrophage activity and atherosclerotic plaque vulnerability.[21] Nevertheless, Ozawa et al. suggested that LGMN had no significant effect on apoptosis of human aortic smooth muscle cells.[24] The above studies indicated that LGMN may affect the development of atherosclerosis by participating in macrophage apoptosis.
Anti-atherogenic effects of legumain
In contrast to these studies, other findings suggest that LGMN may inhibit atherosclerosis. Lunde et al. reported that LGMN not only increased the mRNA expression of the anti-inflammatory IL-10 and the M2 marker CD163, but also decreased the expression of proto-typical pro-inflammatory MCP-1.[68] Also, it is indicated that LGMN could induce anti-inflammatory macrophage polarization by upregulating several genes including CD163, which may reflect monocyte polarization toward the M2 phenotype, while downregulating the M1 phenotype.[68] Furthermore, Gregersen et al. found that transforming growth factor β (TGF-β) may have the ability to stabilize plaques in atherosclerotic lesions.[69]
In summary, LGMN may play diverse roles in the development of atherosclerosis. The stage of pathogenesis and other factors exist in atheroma may contribute to this discrepancy. These conflicting results and potential mechanisms need to be further investigated (Table 1).
Effects of LGMN on atherosclerosis disease.
| Disease | Mechanisms | Action | References |
|---|---|---|---|
| Atherosclerosis | VR (VSMC migration↑, PI3K↑, Akt↑, collagen-3↑, fibronectin↑, elastin↑, monocyte migration↑, TLR4↑, MAPK↑, Smad3↑, and Akt↑) | Promotion | [24, 38–40] |
| Inflammatory response (MCP-1↑, IL-6↑, IL-1β↑, IFN-γ↑, and TNF-α↑) | Promotion | [24, 42–47] | |
| Atherosclerotic plaques (mononuclear macrophages and foam cells) | Promotion | [20, 25, 38, 50–54] | |
| ECM (collagen-1↓, fibronectin↓, pro-MMP-2↑, cathepsin B↑, cathepsin H↑, and cathepsin L↑) | Promotion | [52, 57–60] | |
| Macrophages apoptosis (cathepsin L↑, caspase-3↑, caspase-9↑, Bax↑, LC3↑, and Beclin1↑) | Promotion | [21, 51, 60, 63, 64, 67] | |
| Other mechanisms (IL-10↑, CD163↑, and TGF-β↑) | Inhibition | [68, 69] |
| Disease | Mechanisms | Action | References |
|---|---|---|---|
| Atherosclerosis | VR (VSMC migration↑, PI3K↑, Akt↑, collagen-3↑, fibronectin↑, elastin↑, monocyte migration↑, TLR4↑, MAPK↑, Smad3↑, and Akt↑) | Promotion | [24, 38–40] |
| Inflammatory response (MCP-1↑, IL-6↑, IL-1β↑, IFN-γ↑, and TNF-α↑) | Promotion | [24, 42–47] | |
| Atherosclerotic plaques (mononuclear macrophages and foam cells) | Promotion | [20, 25, 38, 50–54] | |
| ECM (collagen-1↓, fibronectin↓, pro-MMP-2↑, cathepsin B↑, cathepsin H↑, and cathepsin L↑) | Promotion | [52, 57–60] | |
| Macrophages apoptosis (cathepsin L↑, caspase-3↑, caspase-9↑, Bax↑, LC3↑, and Beclin1↑) | Promotion | [21, 51, 60, 63, 64, 67] | |
| Other mechanisms (IL-10↑, CD163↑, and TGF-β↑) | Inhibition | [68, 69] |
Effects of LGMN on atherosclerosis disease.
| Disease | Mechanisms | Action | References |
|---|---|---|---|
| Atherosclerosis | VR (VSMC migration↑, PI3K↑, Akt↑, collagen-3↑, fibronectin↑, elastin↑, monocyte migration↑, TLR4↑, MAPK↑, Smad3↑, and Akt↑) | Promotion | [24, 38–40] |
| Inflammatory response (MCP-1↑, IL-6↑, IL-1β↑, IFN-γ↑, and TNF-α↑) | Promotion | [24, 42–47] | |
| Atherosclerotic plaques (mononuclear macrophages and foam cells) | Promotion | [20, 25, 38, 50–54] | |
| ECM (collagen-1↓, fibronectin↓, pro-MMP-2↑, cathepsin B↑, cathepsin H↑, and cathepsin L↑) | Promotion | [52, 57–60] | |
| Macrophages apoptosis (cathepsin L↑, caspase-3↑, caspase-9↑, Bax↑, LC3↑, and Beclin1↑) | Promotion | [21, 51, 60, 63, 64, 67] | |
| Other mechanisms (IL-10↑, CD163↑, and TGF-β↑) | Inhibition | [68, 69] |
| Disease | Mechanisms | Action | References |
|---|---|---|---|
| Atherosclerosis | VR (VSMC migration↑, PI3K↑, Akt↑, collagen-3↑, fibronectin↑, elastin↑, monocyte migration↑, TLR4↑, MAPK↑, Smad3↑, and Akt↑) | Promotion | [24, 38–40] |
| Inflammatory response (MCP-1↑, IL-6↑, IL-1β↑, IFN-γ↑, and TNF-α↑) | Promotion | [24, 42–47] | |
| Atherosclerotic plaques (mononuclear macrophages and foam cells) | Promotion | [20, 25, 38, 50–54] | |
| ECM (collagen-1↓, fibronectin↓, pro-MMP-2↑, cathepsin B↑, cathepsin H↑, and cathepsin L↑) | Promotion | [52, 57–60] | |
| Macrophages apoptosis (cathepsin L↑, caspase-3↑, caspase-9↑, Bax↑, LC3↑, and Beclin1↑) | Promotion | [21, 51, 60, 63, 64, 67] | |
| Other mechanisms (IL-10↑, CD163↑, and TGF-β↑) | Inhibition | [68, 69] |
Legumain and atherosclerosis treatment
As we all know, statins are the most commonly used lipid-lowering drugs in clinic. They are involved in a variety of inflammatory cytokines. They participate in the immune process of various inflammatory factors and inflammatory pathways, reduce inflammation, affects the composition of lipids in atherosclerotic plaques, improves VR, and thus achieves the goal of stabilizing atherosclerotic plaques. Some of these mechanisms are closely related to regulating LGMN.[20, 54] It has been confirmed that atorvastatin can reduce the expression level of LGMN mRNA in monocytes. Wang et al. demonstrated that LGMN expression gradually decreased with increasing duration of atorvastatin treatment. However, short-term treatment at high doses did not result in a decrease in LGMN expression.[70] The differentiation of monocytes and macrophages was inhibited by atorvastatin, while the expression, activity, and secretion of LGMN were increased.[54] Simvastatin, an 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-CoA) reductase inhibitor, is widely used in the treatment of hypercholesterolemia.[71] Simvastatin affects the activity and expression of LGMN in human myotubes. Among them, simvastatin reduces the processing of pro-LGMN (56 kDa) by inhibiting HMG-CoA reductase, which leads to the decrease of LGMN activity.[72] In addition, the autoactivate of pro-LGMN occurs under acidic pH conditions.[73] Therefore, the inhibition of LGMN activity by simvastatin may be partly due to the impaired transport of pro-LGMN from Golgi to vesicles with acidic pH such as late endosomes or lysosomes. Moreover, the decrease of glucose concentration in cells is also affected by simvastatin. When the glucose concentration is decreased, the pH of lysosomes will increase, resulting in the decrease of pro-LGMN production and expression.[72] Rosuvastatin is the most effective oral lipid-regulating drug. Chen et al. indicated that intensive rosuvastatin therapy reduced serum LGMN concentration and improved left ventricular function without increasing adverse reactions.[74] A clinical trial showed that low-dose colchicine (0.5 mg/day), in addition to statins, was effective in preventing cardiovascular events in patients with stable coronary heart disease.[75] Due to the significant restriction substrate specificity of the cysteine protease LGMN, colchicine is lipophilic and cytotoxic with a low therapeutic index, and is the only known mammalian APE. LGMN is overexpressed in unstable atherosclerotic plaques and therefore activation of prodrugs using LGMN.[57, 60] Smith et al. synthesized the LGMN cleavable peptide sequence, which was subsequently combined with deacetylated colchicine to produce the prodrug in three steps. The drug was more potent in cells with high expression of LGMN compared to cells with low expression of LGMN or cells expressing only the pro-LGMN.[76] Therefore, this new approach is expected to be applied in the targeted therapy of atherosclerotic plaques.
Conclusion and future directions
Atherosclerosis can cause a series of cardiovascular diseases, which is a serious threat to human health and one of the leading causes of death worldwide. Atherosclerosis suffers the effects from LGMN in diverse cell lines and multiple pathways. LGMN is involved in a variety of physiological and pathological processes and has been proven to aggravate atherosclerosis through multiple mechanisms. The pathogenesis of atherosclerosis is very complex and is associated with many factors such as hypertension, hyperlipidemia, diabetes, long-term smoking, and obesity, among which disorders of lipid metabolism have been considered as an independent risk factor for the occurrence and development of atherosclerosis.[77–79] LGMN is a member of the C13 family. LGMN has attracted more and more attention since it was discovered in the 1980s.[25] It is of great significance to investigate whether LGMN promotes atherosclerosis by promoting lipid accumulation. In addition, there are still many questions that need to be addressed in future studies. Treatment with recombinant LGMN significantly inhibited macrophage apoptosis induced by ox-LDL, suggesting that intervention with LGMN may be a new therapeutic target for atherosclerosis.[20, 60] The change of LGMN expression is an important link in initiating or promoting the inflammatory response in carotid atherosclerotic lesions, which may be an important factor leading to the occurrence and development of carotid atherosclerotic lesions. The ideal biomarker must be able to clearly indicate the presence of pathological changes that are essential for the development of As.
Moreover, LGMN has been proposed as a marker for predicting Cardiovascular disease (CVD) prognosis. Since its overexpression is associated with lesion size, plaque vulnerability, and inflammatory markers, which motivates the interest of investigators in monitoring the distribution of LGMN in vivo.[53] Therefore, different imaging strategies have been used, including quenched activity-based probes to visualize active LGMN endopeptidase.[80] Hong et al. synthesized a smart fluorescent-labeled Michael receptor inhibitor (activator protein 1 probe) using the LGMN-specific Ac-hAl-Pro-azaAsn peptide, which only fluoresces after reacting with LGMN. Therefore, it can be used for imaging experiments of macrophages, fibroblasts, neurons, and other living cells.[81] Also, Poreba et al. used a combined HyCoSuL/CoSeSuL approach to develop an LGMN probe (MP-L01) that does not bind to caspases to selectively target this protease.[82] Lunde et al. reported that the mature form of LGMN can be activated by binding to the LGMN selective active probe (ABP) MP-L01.[68] Lunde et al. proved that the level of the plasma LGMN of patients with carotid plaque is higher than that of health control, indicating that LGMN may be a novel biomarker for atherosclerosis.[20] What’s more, serum LGMN levels were found to be positively correlated with levels of High-Sensitive C-Reactive Protein (hs-CRP), a currently recognized inflammatory marker involved in coronary heart disease, further supporting that LGMN could be used as a new biomarker in patients with coronary heart disease.[69] LGMN may be an attractive noninvasive molecular imaging target to determine the extent of disease and prognosis in CVD patients.[83] With the development of selective imaging technology for visualization of active LGMN,[84] the application prospects are promising.
We summarized the structure and distribution of LGMN, and review the mechanisms of its pro-atherosclerotic action in this review. Given that LGMN is widely distributed in various tissues and organs, and is involved in the progression and development of atherosclerosis. The above discussion and summary indicate that LGMN up-regulation is found in the signature events of atherosclerosis. Therefore, LGMN is worth exploring as a potential biomarker. In addition, we emphasized its significant role in atherosclerosis. This may be useful for researchers to develop novel clinical drugs for atherosclerosis by inhibiting or antagonizing LGMN.
Author contributions
These authors contributed equally to this work. Bo-yi Ke, Yu-sheng Qin and Lu Cheng contributed equally to this work.
Conflict of interest statementThe authors declare that there is no conflict of interest.
Funding
This work was supported by the Scientific Research Project of Hunan Provincial Department of Education (20K106), the Project of Hunan Provincial Health Committee (D202302048902) and National Education Ministry College Student Innovation and Entrepreneurship Training Program Project (202210555097), University of South China, China.
