The Dual Role of Low-Density Lipoprotein Receptor-Related Protein 1 in Atherosclerosis

Low-density lipoprotein receptor–related protein-1 (LRP1) is a large endocytic and signaling receptor belonging to the LDL receptor (LDLR) gene family and that is widely expressed in several tissues. LRP1 comprises a large extracellular domain (ECD; 515 kDa, α chain) and a small intracellular domain (ICD; 85 kDa, β chain). The deletion of LRP1 leads to embryonic lethality in mice, revealing a crucial but yet undefined role in embryogenesis and development. LRP1 has been postulated to participate in numerous diverse physiological and pathological processes ranging from plasma lipoprotein homeostasis, atherosclerosis, tumor evolution, and fibrinolysis to neuronal regeneration and survival. Many studies using cultured cells and in vivo animal models have revealed the important roles of LRP1 in vascular remodeling, foam cell biology, inflammation and atherosclerosis. However, its role in atherosclerosis remains controversial. LRP1 not only participates in the removal of atherogenic lipoproteins and proatherogenic ligands in the liver but also mediates the uptake of aggregated LDL to promote the formation of macrophage- and vascular smooth muscle cell (VSMC)-derived foam cells, which causes a prothrombotic transformation of the vascular wall. The dual and opposing roles of LRP1 may also represent an interesting target for atherosclerosis therapeutics. This review highlights the influence of LRP1 during atherosclerosis development, focusing on its dual role in vascular cells and immune cells.


INTRODUCTION
Atherosclerosis (AS), in concert with its related disorders, such as coronary heart diseases, stroke, and peripheral vascular diseases, is the leading cause of morbidity and mortality worldwide (1)(2)(3)(4). Some researchers have characterized atherosclerosis as damage to three major processes: systemic and cellular cholesterol homeostasis, inflammation and apoptosis/endocytosis (5). Atherosclerosis is initiated by endothelial dysfunction due to activation of endothelial cells by irritative stimuli, such as hyperlipidemia, high shear forces, hypertension, and proinflammatory mediators, which allow blood monocytes to permeate the endothelial cell layer and infiltrate into the intima and subintima. After entering into the intima, the monocytes differentiate into macrophages, which subsequently uptake modified low-density lipoprotein (LDL) or oxidized phospholipids to further form foam cells. The successive accumulation of apoptotic foam cells in the endothelium cannot be cleared in time, gradually leading to the formation of thrombi and inflammatory necrotic cores (6,7). Subsequently, the release of the chemoattractant plateletderived growth factor from macrophages, activated platelets and endothelial cells causes vascular smooth muscle cells (VSMCs) to migrate from the medium to the intima, proliferate, undergo apoptosis and senescence, and produce extracellular matrix to form fibrous caps of atherosclerotic plaques to prevent plaques rupture. However, the aberrant proliferation of VSMCs promotes plaque formation, and the balance of VSMC proliferation between migration vs. cell death and senescence determines the population of VSMCs within the atherosclerotic plaques. These processes play important roles in the formation of atherosclerosis and the stability of plaques (8,9). Lastly, the rupture of atherosclerotic plaques promoted by cap thinning due to the death of VSMCs and the breakdown of collagen and ECM leads to thrombosis, potentially resulting in major cardiovascular episodes such as stroke and myocardial infarction (9).
LDL receptor-related protein (LRP1) is a 600-kDa type I glycosylated transmembrane protein belonging to the LDL receptor (LDLR) superfamily and is ubiquitously expressed in multiple cell types (10-13). This multifunctional transmembrane protein has been reported to regulate cholesterol homeostasis, inflammation and apoptosis/endocytosis (14)(15)(16). LRP1 is expressed in both normal and atherosclerotic arteries (17) and can recognize both lipoprotein and non-lipoprotein ligands to participate in a wide variety of biological processes, including lipid metabolism (18), blood-brain barrier (BBB) integrity (19) and macrophage migration (20). Genome-wide association studies have revealed that the LRP1 gene constitutes a susceptibility locus for abdominal aortic aneurysms, elevated plasma lipids and coronary heart disease (21)(22)(23). Many translational studies have shown that LRP1 is involved in two major physiological processes: endocytosis and signaling pathway regulation. The endocytosis of multiple extracellular ligands of LRP1, including apolipoprotein E (ApoE)-and lipoprotein lipase-enriched lipoproteins, thrombospondin, and plasminogen activators, is important in vascular biology and tumor progression. In addition, LRP1 can initiate and regulate diverse signaling pathways, including the mitogen-activated protein kinase (MAPK), insulin receptor (IR), serine/threonine protein kinase (AKT), extracellular signal-regulated kinase (ERK), and c-jun N-terminal kinase (JNK) pathways (24)(25)(26)(27)(28)(29).
In this review, we mainly provide a great detailed discussion of the dual role of LRP1 in regulating atherosclerosis and its implication in antiatherosclerosis or proatherosclerosis, in order to enhance our understanding of the underlying mechanism in order to develop novel prophylactic and therapeutic strategies against cardiovascular diseases (CVDs).

Discovery of LRP1
In 1988, Herz et al. described a cell surface protein containing 4,544 amino acids that was abundant in the liver and had high structural and biochemical similarity with the LDL receptor. They named this protein LDL receptor-related protein (LRP) (12). Later, this receptor was identified by Ashcom et al. and Moestrup and Gliemann, who isolated and sequenced the liver receptor responsible for the catabolism of the α-2-M-proteinase complex (57,58). Beisiegel et al. described LRP as an ApoEbinding protein that plays an important role in cholesterol metabolism by mediating the uptake of LDL from plasma into cells (59). In addition, LRP is a large multifunctional clearance receptor that has been implicated in the hepatic uptake of chylomicron remnants and the removal of both circulation and extracellular space-associated protease-inhibitor complexes (60). The LRP1 gene is located on chromosome 12q13-14 (61) and is synthesized in the endoplasmic reticulum as a transmembrane glycosylated precursor protein with an apparent molecular mass of ∼600 kDa. After it enters the Golgi complex, LRP1 is cleaved to generate two subunits (62). Global knockout of the LRP1 gene in mice is embryonic lethal, demonstrating an essential role for LRP1 in embryogenesis and development (60,63).

Structure of LRP1 and sLRP1
Structure of LRP1 LRP1, also called CD91 or α-2-M receptor, is a type I glycosylated transmembrane protein comprising a large extracellular domain (ECD) (515 kDa, α chain) and a small intracellular domain (ICD; 85 kDa, β chain), which are non-covalently associated on the cell surface. The large ECD contains four clusters of complement-like repeats and epidermal growth factor (EGF) repeats. The small ICD contains one YXxL motif, two dileucine motifs responsible for endocytosis and two NPxY motifs that function as secondary endocytosis signals and binding sites for signaling adaptor proteins (10-12, 18,62,[64][65][66][67][68][69]. The NPxY motif can be phosphorylated on several serine, threonine or tyrosine residues to regulate signal transduction (70)(71)(72). Additionally, LRP1 can undergo intramembrane proteolysis called regulated intramembrane proteolysis (RIP), which induces cleavage of the released ICD of LRP (LRP-ICD) by γ-secretase and subsequent translocation of this domain into the cell nucleus, where it represses interferon-γ promoter activity to suppress inflammation (73,74). Like all members of the LDL receptor family, LRP1 consists of five modular structural units: cysteinerich complement-type repeats (CRs), EGF precursor repeats, βpropeller (YWTD) domains, a transmembrane domain and a cytoplasmic domain (Figure 1) (75). The EGF precursor consists of two cysteine-rich EGF repeats, a YWTD repeat and another EGF-like repeat (76). The extracellular region contains four ligand-binding domains (clusters I-IV) consisting of 2, 8, 10, and 11 CRs, respectively, with clusters II and IV are the major ligand-binding regions (77).

sLRP1: The Soluble Form of LRP1
Similar to a wide variety of receptors and other plasma membrane proteins, LRP1 can be cleaved by cell surface proteases such as hepatic metalloproteinases, tPA and neuronal β-secretase protease (BACE1) to produce soluble LRP1 (sLRP1), which can be detected in plasma and cerebrospinal fluid (19,(78)(79)(80)(81)(82)(83)(84). This process can be accelerated by inflammatory mediators in cultured bone marrow macrophages, such as lipopolysaccharide (LPS) and interferon-γ (IFN-γ); sLRP1 contains the α chain and a 55-kDa fragment of the β chain, suggesting that cleavage occurs near the plasma membrane (85,86). In addition, sLRP1 maintains the ligand-binding properties of cellular LRP1 and may therefore act as a competitive ligand uptake inhibitor that binds LRP1 on the cell surface (87). By binding extracellular ligands interacting with LRP1, sLRP1 could regulate their endocytosis or control multiple cell signaling pathways. In RAW 264.7 macrophagelike cells, sLRP1 was shown to promote tumor necrosis factorα (TNF-α), monocyte chemoattractant protein type-1 (MCP-1)/CCL2 and IL-10 expression through activation of the MAPK and JNK cellular pathways to regulate the inflammatory response (85). These results suggest that sLRP1 may modulate regulatory cytokine expression by macrophages to regulate inflammation or monocyte chemotaxis.
Furthermore, sLRP1 has been demonstrated to be associated with inflammation (85,88,89), and increased levels of circulating sLRP1 have been observed shown to be increased in patients with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) (85). Several studies have also shown that sLRP1 can mediate Aβ clearance from the brain to the bloodstream via transcytosis across the BBB (90)(91)(92), leading to the proposal that sLRP1 could also be used as a biomarker for Alzheimer's disease (93). In addition, the circulating sLRP1 concentration has been reported to be significantly higher in patients with severe hypercholesterolemia than in those with moderate hypercholesterolemia and normocholesterolemic controls. Moreover, as a robust and direct association between sLRP1 and lipid parameters, sLRP1 may be useful as a biomarker for atherosclerosis (94). Another study revealed that sLRP1 is a novel biomarker for the P2Y12 receptor expression, which can aggravate atherosclerosis, in atherosclerotic plaques (95). However, additional studies are needed to confirm the function of sLRP1 in vivo.

THE DUAL ROLES OF LRP1 IN ANIMAL MODELS OF ATHEROSCLEROSIS
Numerous genetic studies have demonstrated the dual roles of LRP1 in atherosclerosis-related cells in different animal models ( Table 3). LRP1 gene deletion promotes the progression of atherosclerosis for progressive plaques, while promotes the regression of atherosclerosis for established plaques. The generation and analysis of animal models with receptor gene deletion have elucidated important functions of LRP1 in lipoprotein metabolism and significantly advanced our understanding of the pathophysiological process in patients with lipid disorder.

LRP1 Gene Deletion Facilitates the Development of Atherosclerotic Lesions
The effect of LRP1 on the progression of atherosclerosis has been tested in ApoE-/-and LDLR-/-mice, with the results showing that atherosclerosis development was enhanced in ApoE-/-, LDLR-/-and ApoE/LDLR double knockout mice harboring LRP

Molecule
Function References

Growth factors
Platelet-derived growth factor (PDGF)-BB, PDGF receptor (PDGFR) β PDGF-BB is a dimeric glycoprotein composed of two B subunits and a major growth factor that binds with high affinity to the cell surface receptor PDGFR β Transforming growth factor-b 1 (TGF-β1) Multifunctional growth factor, involved in interactions with extracellular proteins, cell growth, differentiation and vascular remodeling (121) Transforming growth factor-b 2 (TGF-β2) Multifunctional growth factor, involved in interactions with extracellular proteins, cell growth, differentiation and vascular remodeling (121) Connective tissue growth factor (CTGF; CCN2) Matricellular protein of the extracellular matrix-associated heparin-binding protein family, involved in cell adhesion, migration, and angiogenesis

Blood coagulation
Coagulation factor VIII Blood-clotting protein, participate in blood coagulation (137) Von Willebrand factor (vWF) Adhesive, glycoprotein involved in blood coagulation and wound healing (138)

Others
Annexin VI Member of the calcium-dependent membrane and phospholipid binding (139) proteins; co-receptor of Lrp1, involved in endocytosis processes, interacts with a-2-M CCN1, cysteine-rich angiogenic inducer 61 (CYR61) Secreted, matrix-associated signaling protein involved in apoptosis, adhesion, migration and vascular integrity (141) Decorin (Dcn) Member of the small leucine-rich proteoglycan family that impacts the activities of growth factors, regulates extracellular matrix assembly and cell adhesion (142) Glypican-3: Hedgehog complexes Glypican-3 is a heparan sulfate proteoglycan that impacts embryonic growth by inhibiting the hedgehog signaling pathway Heparan sulfate proteoglycans (HSPGs) Glycoproteins containing one or more covalently attached heparan sulfate chains; present at the cell surface and in the extracellular matrix; endocytic and adhesion receptors, regulate cell migration    (37). Similarly, another study showed that macrophage LRP deletion in the LDLR-deficient mouse model enhanced atherosclerosis development and increased monocyte chemoattractant protein type-1 (MCP-1), TNFα, and matrix metalloproteinase-9 (MMP-9) levels as well as proximal aorta macrophage cellularity (36). Another study showed a similar result that specific deletion of macrophage LRP1 in the ApoE deficient mice increased atherosclerosis, which is concomitant with the accumulation of apoptotic cells and proinflammatory monocytes in lesions (39). Moreover, the inactivation of LRP1 in VSMCs in LDLR-/-mice resulted in disruption of the elastic layer and marked susceptibility to atherosclerosis together with platelet-derived growth factor (PDGF) signaling pathway overactivation (14,121). Furthermore, LRP1 deficiency in macrophages led to an increase in cell death and inflammation and abolished the antiatherosclerotic benefits of the antitumor necrosis factor-α (TNFα) inhibitor adalimumab (63, 168). A similar result was observed in adipocytes, where adipocytespecific LRP1 knockout (adLRP1-/-) mice fed a western diet for 16 weeks exhibited a 3-fold increase in atherosclerosis and enhanced inflammation in adipose tissues compared to adLRP1+/+ mice (98).

LRP1 Gene Deletion Accelerates Atherosclerosis Regression in Mice
In 2018, Paul et al. reported that macrophages (MΦLRP1-/-) can promote atherosclerosis regression independent of plasma lipid levels, increase reverse cholesterol transport (RCT) and cause selective loss of inflammatory M1 macrophages. In this study, ApoE-/-mice were fed a high-fat diet for 12 weeks, and then reconstituted with bone marrow from apoE-producing wildtype (WT) or MΦLRP1-/-mice, then fed a chow diet for 10 weeks. The results showed that MΦLRP1-/-recipients showed 13% smaller plaques, 1.4-fold higher reverse cholesterol transport (RCT), 36% fewer M1 macrophages and 2.5-fold more CCR7+ macrophages in the plaques than those of WT recipients (169).

LRP1 in VSMCs
VSMCs are the primary cell type in the vessel wall and a major component of atherosclerotic plaques at all stages (170,171). VSMCs can also generate extracellular matrix to form the fibrous cap and hence stabilize plaques, yet aberrant VSMCs proliferation promotes atherosclerotic plaque formation (9). The results of some studies indicated that in VSMCs, LRP1 helps to suppress atherosclerosis by inhibiting the platelet-derived growth factor (PDGF) signaling pathway (14,121). However, the opposite result has been observed in other studies, indicating that LRP1 mediates aggLDL uptake to induce high intracellular cholesteryl ester accumulation in VSMCs, causing the formation of VSMCderived foam cells and promoting atherosclerosis progression LDLR −/− /smLRP −/− +HFD smLRP +/+ /LDLR −/− NA Disruption of the elastic layer, SMC proliferation, aneurysm formation, and marked susceptibility to cholesterol-induced atherosclerosis, no effect on plasma cholesterol or triglyceride levels (14) smLRP −/− smLRP +/+ littermates 46 Greater smooth muscle cell proliferation, deficient contractile protein expression, impairment of vascular contractility, and promotion of denudation-induced neointimal hyperplasia Frontiers in Cardiovascular Medicine | www.frontiersin.org (44)(45)(46). Thus, in VSMCs, LRP1 may exert two different and opposing effects on atherosclerosis (Figure 2A).

LRP1 in VSMCs Protects Against Atherosclerosis
VSMCs are induced to proliferate and migrate from the media to the intima, contributing to the development of atherosclerosis and restenosis under pathological conditions (9, 32, 172). The binding of PDGF, a potent mitogen for fibroblasts and VSMCs, to PDGF receptor β (PDGFRβ) activates a signaling pathway that promotes VSMs growth and migration, which are crucial in atherosclerosis and neointima formation and LRP1 has been proven to suppress this process through incompletely understood mechanisms (14,24,33,121,(173)(174)(175). Studies have elegantly demonstrated this effect via the generation of smLRP1-/-mice on a background of LDLR deficiency, showing that smLRP1-/-/LDLR-/-mice are not only more susceptible to cholesterol-induced atherosclerosis than LDLR-/-mice but also exhibit PDGFR overexpression and increased phosphorylation of Smad2, a downstream component of the TGFβ pathway (14,121). Moreover, the PDGF-BB pathway has previously been described as a target of the TGFβ signaling pathway (176)(177)(178). In addition, LRP1 is identical to TGFβ receptor (V), which is a member of the TGFβ receptor superfamily and is expressed on the cell surface together with TGFβ receptors I, II, and III (179). Therefore, the TGFβ activation pathway in the absence of LRP1 could further activate the PDGF-BB signaling pathway by increasing PDGFRβ expression in the arterial wall, promoting the formation of atherosclerotic lesions. Indeed, the vascular pathology observed in smLRP1-/-/LDLR-/-mice was significantly improved by blockade of the PDGF or TGFβ receptor signaling cascades with the tyrosine kinase inhibitor Gleevec (121). Overall, the results of these studies reveal that in VSMCs, LRP1 plays a major role in maintaining the integrity of the vascular wall and reducing atherosclerosis by suppressing the PDGFRβ and TGFβ signaling pathways. The role of the intracellular NPxYxxL motif of the LRP1-ICD in the development of atherosclerosis has attracted increasing attention. One study has thoroughly demonstrated that inactivation of the LRP1 NPxYxxL motif in LDLR-/mice showed a significant 1.5-fold increase in the development of atherosclerosis compared to that observed in LDLR-/control mice. MMP2 activity, which could be degraded by lysosomal proteases after being endocytically taken by LRP1 (113,180), as well as could facilitate VSMC migration and plaque rupture by degrading the extracellular matrix (181,182), showed a significant 2.7-fold increase in the aortas of NPxYxxLinactivated mice. The results of this study also indicated that there was a significant 2-fold increase in the number of apoptotic cells relative to the plaque size in mice with the NPxYxxLinactivation mice, which was caused by the secretion of the proapoptotic cytokine TNF-α. Therefore, we can conclude that the intracellular NPxYxxL motif of the LRP1-ICD is essential for the atheroprotective role of LRP1 (183). LRP1 can also protect against atherosclerosis by regulating the expression of ATPbinding cassette transporter A1 (ABCA1). The results of multiple studies have indicated that increased mitogenic signaling in the absence of LRP1 can regulate ERK1/2 activation, leading to increased cytosolic phospholipase A2 (cPLA2) phosphorylation, which in turn promotes the production of arachidonic acid (14,184,185), a suppressor of LXR-driven ABCA1 expression (186). This cascade reduces cholesterol efflux from VSMCs and promotes the formation of foam cells (187). In summary, LRP1 plays an antiatherosclerotic role in VSMCs, and its absence promotes atherosclerosis development (Figure 2A).

LRP1 in VSMCs Promotes the Development of Atherosclerosis
In addition to protecting against atherosclerosis, many studies have also shown that LRP1 overexpression is associated with atherosclerosis progression in both animal (188) and human models of atherosclerosis (17,97,189). LRP1 can promote the formation of VSMC-derived foam cells, leading to the progression of atherosclerosis. In addition, its expression is upregulated by lipid accumulation during the progression of atherosclerotic lesions in humans, and VSMCs derived from advanced atherosclerotic plaques show higher intracellular lipid deposition than those from less-advanced plaques due to their higher LRP1 expression levels (190). In addition, the results of another study also indicated that the transient receptor potential vanilloid type-1 (TRPV1) activation-induced decrease in LRP1 expression reduces lipid uptake by VSMCs (191). Several studies have shown that LRP1 mediates the uptake of aggLDL and induces adipose differentiation-related protein (ADRP) overexpression, leading to an increase in intracellular lipid deposition in VSMCs. ADRP is localized on the surface monolayer of lipid droplets and is considered a specific marker of lipid droplet formation (41, 42, 44-46, 189, 192, 193). Moreover, aggLDL can further upregulate LRP1 expression in VSMCs in a time-and dose-dependent manner (48). This positive feedback mechanism is highly efficient in promoting the formation of VSMCs foam cells. The formation of VSMC-derived foam cells, a main proatherogenic mechanism of LRP1, is associated with atherosclerotic lesion progression. Therefore, in VSMCs, LRP1 can also promote atherosclerosis development by promoting the formation of foam cells (Figure 2A).

LRP1 in Macrophages
Macrophages are the most abundant type of immune cells in atherosclerotic lesions, playing an important role in all stages of atherosclerosis, from the formation of atherosclerotic lesions to plaque rupture (194). Lipoprotein receptors in macrophages can accelerate the progression of atherosclerosis by facilitating the uptake of atherogenic particles, such as oxidized lipoproteins, and inducing vascular inflammation (172,195,196). However, the role of macrophage LRP1 in atherosclerosis is controversial, as macrophage LRP1 not only protects against but also promotes atherosclerosis ( Figure 2B).

LRP1 in Macrophages Protects Against the Development of Atherosclerosis
Macrophage LRP1 has been shown to exert atheroprotective effects. However, mice macrophage-specific LRP deficiency in an apoE/LDLR double-deficient background did not exhibit altered plasma lipid levels or plasma lipoprotein profiles but FIGURE 2 | Diagram of the mechanism of regulating atherosclerosis of LRP1 in VSMCs and macrophages. (A) LRP1 in VSMCs could degrade extracellular PDFGBB, MMPs, t-PA, and FVIII, which are atherogenic factors, in the way of endocytosis to play the role of anti-atherosclerosis. It could inhibit migration and proliferation of VSMCs to resist atherosclerosis by preventing the activation of TGFβ/Smad2/3/PDGFRβ signaling pathway. Gleevec could also has the effect to inhibit PDGFRβ. Additionally, LRP1 inhibit the activation of ERK1/2 which could suppresses the expression of ABCA1 by promoting phosphorylation of cPLA2. In contrast, LRP1 in VSMCs accelerates the development of atherosclerosis by promoting the uptake of aggLDL and the formation of foam cells. (B) The LRP1 can also be cleaved by γ-secretase to release a 25-kDa β-chain fragment, which translocates to the nucleus to suppress inflammatory gene expression. The phosphorylation of NPxY of LRP1 ICD promotes the expression of ABCA1. Nevertheless, LRP1 in macrophages accelerates the development of atherosclerosis by promoting the uptake of aggLDL and the formation of oxidized LDL. AS, atherosclerosis, ABCA1, ATP-binding cassette transporter A1, cPLA2, cytosolic phospholipase A2. did exhibit an increase in the total atherosclerotic lesion area (37). Additionally, Overton et al. showed that LDLR-/-mice transplanted with bone marrow from macLRP1-/-mice were 40% greater in proximal aorta lesions than that observed in highfat diet-fed mice transplanted with control bone marrow, and accompanied by the increase in proinflammatory factors, such as monocyte chemoattractant protein type-1 (MCP-1) and TNF-α, but en face analysis of the distal aorta showed no significant difference likely attributable to the shor-term experiments (36). Furthermore, atherosclerotic lesions in mice lacking LRP1 expression in macrophages are characterized by increased apoptosis, suggesting that LRP1 prevents atherosclerosis by promoting efferocytosis to remove apoptotic cells from plaques, which is also manifested as inhibition of the p-AKT survival pathway and the promotion of inflammation with increased IL-1, IL-6 and TNF-α expression (63). Furthermore, LDLR-/mice that received lethal irradiation and were reconstituted with bone marrow from MΦLRP1-/-mice fed on a western-type diet for 10 weeks showed increased necrosis and apoptosis, defective efferocytosis and increased inflammation in the lesions (168). Another study showed that the specific deletion of macrophage LRP1 in ApoE-/-mice promoted atherogenesis and apoptotic cell accumulation in lesions, partially due to decreased efferocytosis and increased lesion necrosis compared to ApoE-/mice. In this research, the authors also indicated that 88% more lipid-stainable lesion areas in the proximal aorta and a 138% increase in MOMA-2 stainable intimal macrophages in LDLR-/mice receiving ApoE-/-macLRP1-/-bone marrow compared with those that receiving ApoE-/-marrow. Aorta en face lesions were not significantly different between ApoE-/-macLRP1-/and ApoE-/-BM-recipient mice, which is consistent with the previous report in 2007 (39). In addition, LRP-1 can also affect macrophage polarization and promote polarization toward the anti-inflammatory M2 functional phenotype (197), leading to an increase in the number of anti-inflammatory M2 macrophages in lesions. In another study, LRP1 was proposed to inhibit cellular inflammatory responses in an adipocyte-specific LRP1-deficient mouse model (98). Another potential mechanism by which LRP1 affects inflammation in macrophages may be through the direct regulation of inflammatory gene transcription. In cultured macrophages, the LRP1-ICD can be cleaved from the plasma membrane by γ-secretase upon induction by inflammatory mediators, such as LPS and interferon-γ, and then translocate into the nucleus, where it promotes the nuclear export and proteasomal degradation of interferon regulatory factor 3, thereby limiting the expression of the proinflammatory genes in cultured fibroblasts and macrophages (73,85). Apart from regulating inflammation, a study using a knock-in mouse model of LRP1Y63F, in which the tyrosine in the distal NPxY motif was replaced with phenylalanine to prevent NPxY phosphorylation of LRP1, revealed that LRP1 not only regulates the expression of ABCA1, the major cholesterol exporter, to maintain cholesterol efflux but also integrates cellular cholesterol homeostasis with inflammation and efferocytosis (15). In summary, macrophage LRP1 may primarily exert an atheroprotective effect mainly by decreasing inflammation, facilitating efferocytosis and promoting cholesterol efflux ( Figure 2B).

LRP1 in Macrophages Promotes the Development of Atherosclerosis
Evidence from several in vitro studies shows that LRP1 has proatherogenic properties in macrophages. First, LRP1 is upregulated during macrophage foam cell formation (198).
Second, macrophage LRP1 has been shown to play a vital role in the translocation of 12/15-lipoxygenase, promoting the formation of oxidized LDL (199,200). Third, LRP1 was shown to mediate the internalization of aggLDL and apoE-rich atherogenic lipoproteins, along with LDLR, into macrophages (46,201,202). Through the above mechanisms, the accumulation of lipids and macrophage-derived foam cells increases, leading to the progression of atherosclerosis. In addition, the results from an animal study proved that mice with macrophages lacking LRP1 (MΦLRP1-/-mice) exhibit accelerated regression of atherosclerosis and enhanced reverse cholesterol transport (RCT), and drive macrophage egress from lesions by inducing expression of the motility receptor CCR7 (169). Therefore, macrophage LRP1 can also lead to atherosclerosis progression by promoting foam cell formation ( Figure 2B).

LRP1 in Endothelial Cells
Atherosclerosis is a chronic process initiated by endothelial dysfunction and structural changes (203). Although LRP1 is highly expressed in a variety of cells, its protein expression levels in endothelial cells are low (96,204). However, LRP1 expression is tightly regulated by various physiological conditions in endothelial cells, reflecting its crucial role in these cells. An elegant study showed that LRP1 regulates hypoxiamediated angiogenesis by inhibiting PARP-1 activity and suppresses endothelial cell proliferation by preventing cell cycle progression in an oxygen-induced retinopathy (OIR) mouse model (205). Another study also showed that LRP1 can mediate bone morphogenetic protein-binding endothelial regulator (BMPER)-mediated bone morphogenetic protein 4 (BMP4) signaling to regulate endothelial cell migration and angiogenesis (206). Angiogenesis is the process of growing new blood vessels from the existing vascular network that occurs during embryonic development and throughout adulthood, and begins under wound healing and pathological conditions, such as retinopathy. Pathological retinal angiogenesis produces physiologically defective blood vessels, leading to exudation and hemorrhage that threatens vision (207). Moreover, a study showed that LRP1-dependent BMPER signaling is required for LPS-induced nuclear factor of activated T cells 1 (NFATc1) activation to induce acute inflammatory responses in endothelial cells (208), which may cause the initiation of atherosclerosis. Interestingly, LRP1 can also promote peroxisome proliferatoractivated receptor-γ (PPARγ) activity by acting as a coactivator to regulate lipid and glucose metabolism in endothelial cells (209). Therefore, it can be used to treat pathological retinal angiogenesis induced by diabetic retinopathy and atherosclerosis by regulating the expression and function of LRP1 in endothelial cells.

LRP1 in Adipocytes
Another cell type with high LRP1 expression and associated with the development of atherosclerosis is adipocytes. An increasing number of studies have shown that adipose tissues, especially those in the perivascular area surrounding the vessel wall, such as the aorta, coronary artery, and carotid artery, also play a vital role in the pathogenesis of atherosclerosis (210,211). Perivascular adipose tissue (PVAT) is a unique conglomerate of various cell types, including adipocytes, preadipocytes, and mesenchymal stem cells that are embedded in a matrix that is invested with microvessels, and that is important for the maintenance of the vascular structure and the regulation of vascular function and homeostasis (210). The interaction between perivascular adipocytes and vascular wall cells, such as endothelial cells and VSMCs, is essential for normal vascular function, and may be disturbed in diseases such as atherosclerosis. In the early process of hyperlipidemia, atherosclerosis prone animal models and human arteries, PVAT expansion and the production of chemokines near the adventitia of large arteries have been detected, leading to the aggravation of inflammation, which may play a fundamental role in the pathogenesis of the cardiovascular disease (CVD) (212). Adipose tissues have been shown to exhibit increased inflammation with enhanced monocyte-macrophage infiltration and inflammatory gene expression in mice with adipocyte-specific LRP1 knockout (adLRP1-/-) in PVAT, and that mice transplanted with PVAT from adLRP1-/-mice displayed a 3-fold increase in atherosclerosis compared to those transplanted with PVAT from adLRP1+/+ mice after a western diet (98). In addition, mice with LRP1 knocked out in adipocytes (adLRP1-/-mice) exhibited delayed postprandial lipid clearance, reduced fat stores, improved glucose tolerance and decreased body weight compared to wild-type mice (213). Taken together, the results of these studies have added the adipose tissue to the list of anatomic sites where LRP1 expression is important for atheroprotection. Thus, it is possible to prevent the development of atherosclerosis and relieve associated clinical symptoms by regulating LRP1 in adipocytes, including through the use of natural and synthetic LRP1 agonists.

LRP1 in Neutrophils
LRP1 is also abundantly present in neutrophils, which are the most abundant type of white blood cell in the human circulation and the principal cell type during acute inflammatory reactions (97,214). The number of circulating neutrophils is a predictor of future adverse cardiovascular events and positively correlates with the size of developing lesions in humans and mice, respectively (215,216). The expression of endothelial cell adhesion molecules (e.g., E-selectin, Pselectin, and intercellular adhesion molecule-1) increases once the endothelial cell dysfunction is triggered by exposure to irritative stimuli such as hyperlipidemia and proinflammatory cytokines, which then triggers the recruitment of neutrophils. In addition, neutrophils can release chemotactic proteins and proinflammatory mediators to promote monocyte recruitment as well as vascular inflammation, promoting atherosclerosis development. Furthermore, neutrophils can degranulate large amounts of different proteases including matrix metalloproteases (MMPs), myeloperoxidase (MPO) and neutrophil elastase or form neutrophil extracellular traps, leading to a thinner fibrous cap and subsquent plaque rupture (214,217). The results of one study indicated LRP1 blockade could prevent intravascular adherence and neutrophil recruitment within the ischemic tissue induced by PAI-1 derived from both leukocytes and nonleukocytic sources (158). Another similar study showed that LRP1 synthesized and expressed by neutrophils accounts for the r-tPA-induced migration and degranulation of neutrophils (217), which can aggravate tissue damage and inflammation. Additionally, in a hind limb ischemia model, LRP-1 was observed to act as the receptor of cytokine midkine (MK) to support neutrophil adhesion and trafficking by promoting the highaffinity conformation of β2 integrin, suggesting a role of LRP-1 in acute inflammation (218). Furthermore, another study also demonstrated that LRP1 can bind to β2 integrin complex expressed in neutrophils to regulate the firm adhesion and subsequent transmigration of neutrophils (100). Taken together, the result of these studies indicate that LRP1 in neutrophils primarily plays a role in neutrophil-associated inflammation.

LRP1 in T Cells
T lymphocytes account for another majority of immune cells in human atherosclerotic plaques obtained from endarterectomy (219). A large body of evidence from animal studies suggests that the T-cell response is proatherogenic. Apoe-/-mice crossed with mice lacking the V(D)J recombination-activating protein 1 Rag1 (Rag1-/-) or mice with a severe combined immunodeficiency (SCID) mutation (scid/scid mice) are immunodeficient owing to impaired T-cell and B-cell development and showed reduced atherosclerosis lesions when fed a chow diet (220). Poor LRP1 expression in T cells led to suppression of T cell adhesion to fibronectin and ICAM-1 as well as TCR-induced activation, subsequently suppressing accumulation at sites of inflammation (221). Nevertheless, another study showed that LRP1 inhibites the adhesion of T cells to ICAM-1 and fibronectin via JAK signaling (222). Furthermore, LRP1 on T cells interactes with membrane-associated proteinase 3 (mP3) on neutrophils resulting in inhibition of the inflammatory response (101). In summary, LRP1 on T cells may also play a major role in the inflammatory response associated with T cells.

CONCLUSIONS
LRP1 is a large, multifunctional type 1 transmembrane receptor and essential for maintaining basic cellular functions and the development of organisms. Due to its ability to mediate actions of a broad range of ligands, LRP1 participates in the development of multiple degenerative diseases, such as atherosclerosis and Alzheimer's disease. In this review, we summarized the dual and opposing roles of LRP1 in atherosclerosis in vivo and in vitro. The role of LRP1 in VSMCs and macrophages in the development of atherosclerosis is different and opposite in vivo and in vitro, which may be the reason that the complexity of cross-talks among various signal pathways and different cell types and organ systems in vivo. Probably more surprising, for established plaque, the lack of LRP1 expression in macrophages unexpectedly promotes atherosclerosis regression. As a result, the opposite effect of LRP1 involved in the regulation of atherosclerosis might depend on whether the plaque is growing or shrinking. The dual role of LRP1 in mediating the effect of TNF-α on vascular inflammation and in balancing the effects of CD47 on efferocytosis could be involved in this phenomenon (223,224). However, in endothelial cells and adipocytes, LRP1 may promote resistance to atherosclerosis, primarily by inhibiting inflammation. These results suggest that the functionality of LRP1 is dependent on the cell type in which this receptor is expressed.

Therapeutic Considerations
This review may provide a basis for the development of new therapeutic approaches for atherosclerosis that target LRP1 and its downstream cellular signaling pathways. For example, the Cterminal half of the cluster II CR9 domain (Gly1127-Cys1140) has been reported to be crucial for LRP1-mediated aggLDL binding and internalization in human VSMCs (hVSMCs) (45). Moreover, anti-P3 (Gly1127-Cys1140) antibodies (Abs) that specifically block the LRP1 (CR9) domain have been shown to efficiently prevent LRP1-mediated aggLDL internalization and aggLDL-induced LRP1 upregulation to prevent human macrophages and VSMCs from forming foam cell formation (45,225). These findings indicate that this strategy could be used to prevent the occurrence and progression of atherosclerosis. In terms of established plaques, inhibiting LRP1 in macrophages with blocking antibodies could accelerate plaque regression, potentially alleviating atherosclerosis and related cardiovascular and cerebrovascular complications. Notably, LRP1 can regulate the endocytic clearance of several MMPs (113,226), which could degrade the extracellular matrix to promote VSMC migration and thin the fibrous cap, causing plaque rupture and leading to myocardial infarction and stroke (227). Therefore, whether the inhibition of LRP1 would disrupt the signaling pathways involved in the proliferation and migration of VSMCs and proteolysis activity of MMPs will require a more comprehensive understanding.
However, some studies have shown that the natural LRP1 agonists SERPINs, such as α-2-macroglobulin (A2MG), α-1antitrypsin (AAT), antithrombin III, and synthetic LRP1 agonists (SP16) can be used to treat myocardial ischemia-reperfusion injury by inducing cytoprotective signals in cardiomyocytes, such as the activation of Akt-and ERK1/2-dependent prosurvival as well as anti-inflammatory signaling pathways. Moreover, AAT and SP16 are under clinical development, and also there are no treatment-related serious adverse events or toxicity (228,229). Thus, the natural and synthetic agonists of LRP1 may be useful in treating atherosclerosis by inhibiting inflammation and promoting vascular cell survival, but additional evidence is needed to elucidate the associated mechanism. Gene therapy could be used to increase LRP1 expression. For instance, it may be possible to use adeno-associated virus-2 (AAV-2) carrying the cDNA of LRP1 or its smaller fragments to increase LRP1 expression in vascular cells to resist atherosclerosis. In a mouse model of Alzheimer's disease, treatment with recombinant ligand-binding domain IV of LRP1 by using an in situ arterial brain perfusion technique for 3 months reduced brain Aβ levels (90). Therefore, gene therapy and recombinant LRP1 could be used to treat and prevent atherosclerosis.
In summary, in this review, LRP1 was shown to participate in a large number of physiological activities as a coreceptor and to interact with many adaptor proteins through its cytoplasmic domain. The understanding of the mechanisms and the further identification of LRP1 partners may open up new ways to treat metabolic diseases, such as lipid metabolism, atherosclerosis, inflammation, Alzheimer's disease and obesity. Futher investigations will most likely uncover even more functions of these receptor beyond those considered here.

AUTHOR CONTRIBUTIONS
LM and BH designed the review. JC, YS, and SP wrote the manuscript. All authors contributed to the article and approved the submitted version.