Low-Density Lipoprotein Receptor – Related Protein-1 Role in the Regulation of Vascular Integrity

The low-density lipoprotein receptor–related protein-1 (LRP1) is a large endocytic receptor that was identified originally when Ashcom et al and Moestrup et al isolated and sequenced the liver receptor responsible for catabolism of α 2 macroglobulin:proteinase complexes and Herz et al cloned a large protein containing multiple LDL receptor type A repeats (LDLa) and demonstrated its role in chylomicron remnant uptake. LRP1 is highly expressed in vascular smooth muscle cells (SMCs), neurons, macrophages, and fibroblasts but only expressed at very low levels in endothelial cells. In addition to its endocytic function, LRP1 has also been found to modulate signaling pathways and to regulate several important physiological processes. Genome-wide association studies reveal that the LRP1 gene represents a susceptibility locus for abdominal aortic aneurysms, as well as for elevated plasma lipids and coronary heart disease. LRP1 regulates important physiological processes including blood–brain barrier integrity and macrophage migration. Studies in mice reveal that LRP1 expressed in vascular SMCs or macrophages protects the vasculature from the development of atherosclerosis (see below). Deletion of the lrp1 gene in mice results in embryonic lethality, revealing a critical role during development.

T he low-density lipoprotein receptor-related protein-1 (LRP1) is a large endocytic receptor that was identified originally when Ashcom et al 1 and Moestrup et al 2 isolated and sequenced the liver receptor responsible for catabolism of α 2macroglobulin:proteinase complexes 3,4 and Herz et al 5 cloned a large protein containing multiple LDL receptor type A repeats (LDLa) and demonstrated its role in chylomicron remnant uptake. 6,7LRP1 is highly expressed in vascular smooth muscle cells (SMCs), neurons, macrophages, and fibroblasts but only expressed at very low levels in endothelial cells.In addition to its endocytic function, LRP1 has also been found to modulate signaling pathways and to regulate several important physiological processes.Genome-wide association studies reveal that the LRP1 gene represents a susceptibility locus for abdominal aortic aneurysms, 8 as well as for elevated plasma lipids 9 and coronary heart disease. 10LRP1 regulates important physiological processes including blood-brain barrier integrity [11][12][13] and macrophage migration. 14,15Studies in mice reveal that LRP1 expressed in vascular SMCs [16][17][18][19] or macrophages [20][21][22] protects the vasculature from the development of atherosclerosis (see below).Deletion of the lrp1 gene in mice results in embryonic lethality, revealing a critical role during development. 23,24

See Insight into Dudley K. Strickland on page 498
During the isolation of LRP1, a molecule termed the receptor-associated protein (RAP) was identified when it copurified with LRP1. 1,25RAP binds with high affinity to LRP1, 26 as well as other members of the LDL receptor family, 27,28 and blocks the binding of ligands to these receptors. 26,291][32] This review will describe the mode of ligand recognition by LRP1 and its role in regulating thrombosis and in maintaining the integrity of the vasculature.

Canonical Mode of Ligand Recognition by LRP1
LRP1 is composed of a modular structure consisting of clusters of LDLa repeats where most of the ligands bind (clusters I-IV; Figure 1A).In addition, LRP1 contains epidermal growth factor-like repeats, β-propeller domains, a transmembrane domain, and an intracellular domain (ICD; Figure 1A).LRP1 binds >30 distinct ligands that are structurally unrelated.How LRP1 recognizes so many different molecules has raised questions on the mechanisms by which ligands interact with this receptor.Answers to these questions have come from structural studies that have identified a canonical mode © 2014 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.orgDOI: 10.1161/ATVBAHA.113.301924 Abstract-Low-density lipoprotein receptor-related protein-1 (LRP1) is a large endocytic and signaling receptor that is widely expressed.In the liver, LRP1 plays an important role in regulating the plasma levels of blood coagulation factor VIII (fVIII) by mediating its uptake and subsequent degradation.fVIII is a key plasma protein that is deficient in hemophilia A and circulates in complex with von Willebrand factor.Because von Willebrand factor blocks binding of fVIII to LRP1, questions remain on the molecular mechanisms by which LRP1 removes fVIII from the circulation.LRP1 also regulates cell surface levels of tissue factor, a component of the extrinsic blood coagulation pathway.This occurs when tissue factor pathway inhibitor bridges the fVII/tissue factor complex to LRP1, resulting in rapid LRP1-mediated internalization and downregulation of coagulant activity.In the vasculature LRP1 also plays protective role from the development of aneurysms.Mice in which the lrp1 gene is selectively deleted in vascular smooth muscle cells develop a phenotype similar to the progression of aneurysm formation in human patient, revealing that these mice are ideal for investigating molecular mechanisms associated with aneurysm formation.Studies suggest that LRP1 protects against elastin fiber fragmentation by reducing excess protease activity in the vessel wall.These proteases include high-temperature requirement factor A1, matrix metalloproteinase 2, matrix metalloproteinase-9, and membrane associated type 1-matrix metalloproteinase.In addition, LRP1 regulates matrix deposition, in part, by modulating levels of connective tissue growth factor.Defining pathways modulated by LRP1 that lead to aneurysm formation and defining its role in thrombosis may allow for more effective intervention in patients.(Arterioscler Thromb Vasc Biol.2014;34:487-498.) of ligand binding by LRP1 and other members of the LDL receptor family.Ligand recognition occurs when an ε-amino group of a specific lysine residue located on the ligand forms salt bridges with carboxylates of aspartate residues within the LDLa repeats.These aspartic acid residues form an acidic pocket that is constrained by the presence of a calcium ion to which they are coordinated (Figure 1B and 1C).The interaction with ligand is strengthened by an aromatic residue that forms van der Walls interactions with the aliphatic portion of the lysine residue that is docked in the acidic pocket.Often, a second lysine residue is present that can form weak electrostatic interactions with additional acidic residues on the LDLa repeat.][35][36][37][38][39][40] One surprise of this model is the small contact interface between the LDLa repeat and the region surrounding the primary lysine residue.For example, in the case of the receptor associated protein D3 domain/LDL structure, the contact interface between ligand and receptor around lysine 256 is relatively small (696 Å 2 ). 37Similar results have been obtained for the contact interface other ligand receptor structures (eg, apoE receptor 2 fragment with the R5-6 fragment of reelin). 34r comparison, most high-affinity protein interactions have an interface in which the total area buried by the components in the recognition site is in the order of 1600 Å 2 . 41Thus, for LRP1 (and other LDL receptor family members) to bind their ligands with high affinity, avidity effects must contribute to the interaction in which multiple lysine residues on the ligand interact with distinct LDLa repeats on the receptor.

Endocytic Role of LRP1: Factor VIII and von Willebrand Factor Catabolism
LRP1 is abundantly expressed in the liver, where one of its major functions is to mediate the endocytosis and subsequent degradation of certain lipid particles and proteins found in the circulation.One of these proteins is coagulation factor VIII (fVIII) which along with factor IX (fIX) are key plasma proteins that are deficient in the bleeding disorders hemophilia A and hemophilia B, respectively.fVIII is an inactive cofactor that circulates in complex with von Willebrand factor (vWf). [42][43][44] On injury within the vasculature, fVIII is activated to enzymatically active fVIII by limited proteolysis resulting in its dissociation from vWf.Enzymatically active fVIII then assembles on the surface of cells, such as platelets, 45 endothelial cells, 46 and macrophages 47,48 along with enzymatically active fIX to form the macromolecular Xase complex.This complex activates factor X, the next proenzyme in the coagulation cascade.In the circulation, fVIII levels are regulated not only by its biosynthesis but also by hepatic receptors that mediate its clearance.
The identification of LRP1 as a hepatic receptor responsible for the catabolism of fVIII was reported by 2 groups who demonstrated a direct interaction between fVIII and LRP1. 49,50urther, these studies demonstrated that LRP1-expressing cells, but not LRP1-deficient cells, mediate the cellular catabolism of 125I-labeled fVIII.The physiological relevance of this interaction was demonstrated when the in vivo clearance of 125I-labeled fVIII, injected as a complex with vWf, was significantly delayed by coinjection of RAP with fVIII. 49,51hese initial results were confirmed by crossing LRP1 flox/flox with MXIcre mice to generate a hepatic deletion of LRP1.This resulted in a 2-fold increase in plasma fVIII levels and a prolongation in clearance of 125 I-labeled fVIII when injected into the plasma. 52Curiously, the levels of vWf were also elevated in these mice.
Additional studies have revealed that LRP1 is not the only hepatic receptor that is involved in the clearance of fVIII.This was first detected by observing that plasma levels of fVIII increased in MXIcre/LRP1 flox/flox mice after the administration of RAP, 52 revealing that a second RAP-sensitive mechanism exists for fVIII clearance.The identity of the second receptor was confirmed using LDL receptor (LDLR)-deficient mice.The data revealed that although LDLR deficiency alone had no impact on circulating fVIII levels, when both LDLR and LRP1 were genetically deleted in mice, the fVIII levels were even higher than those in LRP1-deficient animals alone. 53hus, both LRP1 and LDLR cooperate in regulating fVIII levels and clearance in vivo, with LRP1 playing a more prominent role in this process than LDLR.
Several of the molecular details of LRP1-mediated fVIII catabolism are not fully understood, and some questions remain at this time.First, vWf is known to delay the clearance of fVIII.Thus, in vWf-deficient mice the levels of fVIII are lower than normal but can be restored to normal levels with injection of vWf. 54Likewise, in patients with von Willebrand disease, fVIII levels are decreased but this can be corrected by an infusion of vWf. 55,56Because plasma fVIII binds tightly to vWf, which is in large molar excess over fVIII, and because vWf blocks the binding of fVIII to LRP1, 50 questions exits on how LRP1-mediated uptake of fVIII occurs.LRP1 undergoes very rapid and constitutive endocytosis; therefore, one possibility is that LRP1 binds and recognizes free fVIII generated when it dissociates from the fVIII/vWf complex.Once bound to LRP1, fVIII is rapidly removed by endocytosis leading to its degradation.This process drives the equilibrium toward free fVIII because its binding to LRP1 results in an irreversible process (ie, internalization and degradation; Figure 2).It is also possible that because heparan sulfate proteoglycans facilitate the clearance of fVIII, 51 heparan sulfate proteoglycans in the liver might promote this process by concentrating the fVIII/ vWf complex on the cell surface and perhaps even expedite the dissociation of vWf from fVIII by increasing the dissociation rate (Figure 2).Similar mechanisms (ie, sequestering on heparan sulfate proteoglycans and transfer to LRP1) have been proposed for the LRP1-mediated uptake of chylomicron remnants. 57This proposal is supported by findings that fVIII effectively binds to the surface of LRP1-expressing cells via interactions with heparan sulfate proteoglycans when presented as a complex with vWf, 51 and by finding that 125I-labeled fVIII is selectively removed from the fVIII/vWf complex in cells expressing LRP1. 49A second question that needs resolving is the contribution that fVIII activation plays in its in vivo clearance.This question arises from the fact that activation of fVIII results in its release from vWf while at the same time exposing a high-affinity site on the A2 domain that is recognized by LRP1, 58,59 suggesting that activated forms of fVIII might be a preferred ligand for LRP1-mediated clearance.
Recent evidence suggests that LRP1 may also participate in the clearance of vWf.A complex relationship exists between vWf and LRP1 because no direct binding between vWf and LRP1 has been detected using in vitro binding assays. 50In contrast, vWf levels are reported to increase when LRP1 is genetically deleted in the liver. 52The major site of vWf clearance seems to be the liver, 61 where hepatic macrophages play a major role in this process. 62Recently, Rastegarlari et al 63 examined the clearance of vWf in mice containing a conditional deletion of macrophage LRP1 (macLRP1-) and observed a significant delay in its clearance.Further, the study noted that vWf bound directly to LRP1 in a RAP-sensitive manner, but only under conditions of high shear where vWf changes conformation and exposes domains that are normally enclosed in its globular form. 64,65

Regulation of Cell Surface Proteolytic Activity by LRP1
LRP1 has the potential to modulate cellular-mediated proteolysis by regulating surface levels of receptors involved in these processes.One of these cellular receptors is tissue factor (TF), a component of the extrinsic blood coagulation pathway. 66his pathway is initiated when factor VII binds TF, which then activates fX to fXa leading to thrombin generation.The pathway is regulated by a potent inhibitor, TF pathway inhibitor, Figure 2. Model for the role of low-density lipoprotein receptor-related protein-1 (LRP1) in the hepatic catabolism of factor VIII (fVIII).In this model, LRP1 binds fVIII that has dissociated from von Willebrand factor (vWf).The kinetics of dissociation of fVIII from vWf has been reported 60 and at 35°C occurs with a t 1/2 of 100 s.Because LRP1 mediates the rapid endocytosis of ligands, this may drive the equilibrium process to generate more free fVIII.fVIII also binds to heparan sulfate proteoglycans (HSPG) on the liver surface.This binding may slightly accelerate the dissociation of vWf-fVIII complex on the cell surface although this has not been demonstrated.It has been demonstrated that 125I-labeled fVIII is selectively removed from the vWf-fVIII/complex and internalized in cells expressing LRP1, even in the presence of a large excess of vWf. 49hich forms a tight complex with both fXa and fVIIa.TF pathway inhibitor contains 3 Kunitz-type protease inhibitor domains, the first 2 of which form a tight complex with fVIIa and Xa and inhibit their proteolytic activity. 67Interestingly, the C-terminal portion of TF pathway inhibitor interacts with LRP1, 68 which bridges the fVII/TF complex to LRP1.This results in internalization of the fVIIa/fXa/TF complex and downregulation of TF-mediated coagulant activity. 691][72] Binding of urokinase-type plasminogen activator to urokinase-type plasminogen activator receptor provides a cell-based proteolytic pathway that is known to stimulate activation of signaling pathways, and the role of LRP1 in regulating this pathway has been reviewed recently. 73

Signaling Roles of LRP1
In addition to its role in transporting molecules into cells for degradation, LRP1 is known to modulate and participate in signaling pathways, and several of these pathways have been reviewed recently. 74There are multiple ways that LRP1 can accomplish this feat.First is the classic role of extracellular ligand binding to LRP1, which in turn orchestrates the endocytosis of ligands and their subsequent trafficking to lysosomes for degradation.Several molecules involved in signaling pathways have been identified to bind to LRP1 (Table ), and thus LRP1 may modulate these pathways by binding and removing the ligand.Second, in certain cells, it seems that the association of a ligand with LRP1 initiates a signaling response (Table ).The mechanisms of how this occurs are still under investigation but likely implicate the association of various adaptor molecules involved in signaling with the cytoplasmic domain of LRP1 via interaction with the NPxY motifs in this receptor.Third, the ICD of LRP1 can interact with several adaptor proteins that are involved in signaling pathways to form a signaling complex.For example, tissue-type plasminogen activator activates the N-methyl-D-aspartate receptor cascade via an LRP1-mediated interaction with the adaptor protein postsynaptic density protein 95, which links the N-methyl-D-aspartate receptor and LRP1. 75Finally, LRP1 is known to undergo regulated intramembrane proteolysis in which shedding of the LRP1 ectodomain generates a substrate for γ-secretase-mediated cleavage of LRP1, which releases the ICD.The ICD of LRP1 then translocates to the nucleus and modulates gene expression (see below). 76

Role of LRP1 in SMC Biology
In healthy vessels, SMCs are in a contractile phenotype and respond to changes in pulse pressure by contracting.5][106] Recent studies in mice in which the lrp1 gene has been deleted selectively in vascular SMCs (smLRP1-) reveal that LRP1 modulates the process of SMC phenotypic switching.Thus, transmission electron microscopy analyses of the aortic vessel wall demonstrated that SMCs within vessels from smLRP1-mice seem to be in a more synthetic phenotype in adult aorta (Figure 3). 19hese SMCs may have more synthetic organelles and have fewer focal adhesions (FA), consistent with a synthetic SMC phenotype.This hypothesis is further supported by studies demonstrating that aortic SMCs isolated from smLRP1-mice proliferate more rapidly and migrate faster than those isolated from sibling control mice. 18ome clues as to potential mechanisms by which LRP1 mediates SMC phenotypic switching are given by the ability of this receptor to affect cell-cell and cell-matrix interactions.Cell adhesion and deadhesion are important in tissue remodeling during development, wound healing, and cell proliferation.During these processes cells change from a strongly adherent state to a state of weaker adherence.This involves restructuring of actin stress fibers and FA while maintaining the spread cell shape and is mediated by matricellular proteins.LRP1 is involved in adhesion and deadhesion of cells through regulating integrin and FA interactions 15,91,107 and facilitates cell migration by modulating the detachment of cells at the trailing edge of the cell by mediating internalization of adhesion complexes containing integrins. 15LRP1 is also involved in FA disassembly, leading to cell detachment, 91 and associates with CD44 108 or with integrin α M β 2 14 to regulate cell detachment.In addition to these functions, LRP1 regulates the cellular trafficking and transport of β1-integrin to the cell surface 109 and also mediates β1-integrin recruitment and stimulation of β1-integrin-linked kinase. 110Integrin-linked kinase is a serine-threonine kinase which is essential for the formation of stress fibers and FA and, therefore, is known to strengthen integrin-cytoskeleton connections.Together, these studies identify attractive mechanisms for the role of LRP1 in regulation of SMC phenotype by modulating cellular adhesion and deadhesion processes.

LRP1 Protects Against Aneurysm Formation
Vessel wall homeostasis is established by the intricate work of multiple proteins, which provide the capacity to endure the hemodynamic forces of blood pressure and to respond to injury appropriately.The vascular structure and composition are established by 2 major extracellular matrix (ECM) components, elastin and collagen fibers, produced by vascular SMCs. 111Medial elastin fibers are woven into an interconnecting laminar network, called the elastic lamina (EL).The EL is designed to bear the load of cardiac cycle and transfer stress throughout the vessel wall. 112Functional failure of these ECM molecules causes several cardiovascular diseases manifested by abnormal matrix deposition, as well as phenotypic alterations of vascular SMCs.One group of diseases is aortic aneurysms and aortic dissections, which account for 1% to 2% of all deaths in Western countries 113 and are usually asymptomatic until they rupture which most often result in death.Clinicians recognize 2 forms of aortic aneurysms.The most common form, abdominal aortic aneurysms, is typically associated with risk factors such as advanced age and smoking.Electron microscopy images of sibling control mice expressing low-density lipoprotein receptor-related protein-1 (LRP1; A and C) and smLRP1-mice (B and D) visualizing the intact (A) vs disrupted (B) lamella (*) including the internal elastic lamina (IEL).Compare higher magnification (C) of vascular SMCs in contact with organized extracellular matrix (ECM) with collagen fiber (col) in aorta of control mice with SMCs loosely in contact with unorganized collagen in aortas from smLRP1-mice (D).Medial SMCs in control aorta are contractile with classical hill-and-valley morphology (C).SMCs from smLRP1mice have prominent synthetic organelles (D, bracket).Proteolytic products of ECM in smLRP1-mice are cleared by phagocytosis by a macrophage (Mϕ; D; bar=2 µm for all images).Adapted with permission from Muratoglu et al. 19 Authorization for this adaptation has been obtained both from the owner of the copyright in the original work and from the owner of copyright in the translation or adaptation.March 2014 The second form of the disease, thoracic aortic aneurysms, occurs in all age groups and is more highly associated with genetic factors. 106Unfortunately, our current understanding of the molecular mechanisms leading to aneurysm formation is limited.Pathologically, inherited forms of thoracic aortic aneurysms typically show destructive matrix remodeling with elastin fragmentation and proliferation of vascular SMCs. 113enetic studies have identified several genes that directly contribute to thoracic aortic aneurysms, and these studies have revealed that perturbed extracellular matrix signaling cascade interactions and deficiencies in SMC contraction represent key mechanisms that contribute to this disease. 106Thus, heterozygous mutations in α-smooth muscle actin, encoded by ACTA2 gene, are associated with thoracic aortic aneurysms and acute aortic dissections. 104,105enetic studies reveal that LRP1 expressed in SMCs contributes to the integrity of the vessel wall [16][17][18][19]114 and protects against aneurysm formation. Thus mice with a SMC-specific knockout of LRP1 (smLRP1-) mice have significant and extensive aortic dilatation which is attributed to extensive disruption of EL with numerous breaks.16,19 Interestingly, this effect does not seem to arise from excessive platelet-derived growth factor (PDGF) signaling, as the PDGF signaling pathway does not appear to be abnormal in smLRP1-mice in the absence of LDL receptor deficiency.19 Rather, the extensive disruption of the EL is smLRP1-mice is likely the result of excessive expression of several proteases.A newly discovered LRP1 ligand, high-temperature requirement factor A1 (HtrA1), is one of these proteases that is present in excess in the aortic wall of smLRP1-mice.19 HtrA1 is a secreted protease that degrades several ECM molecules and is implicated in age-related macular degeneration confirming a role in matrix degradation.115 In addition to HtrA1, increased levels of matrix metalloproteinase-9, matrix metalloproteinase 2, and membrane associated type 1-matrix metalloproteinase are also present in the vessel wall of smLRP1-mice, 19 all of which contribute to degradation of elastic fibers and the development of aneurysms in mouse models.19 This is attributed to excessive connective tissue growth factor accumulation in vessels of these mice.19 Connective tissue growth factor is a member of the cysteinerich angiogenic inducer 61/connective tissue growth factor/ nephroblastoma overexpressed (CCN) family of secreted matricellular proteins that is a key mediator of fibrosis and plays an important role in vascular development.118 Together, these studies highlight the important role for LRP1 in vascular homeostasis, in which it protects the integrity and function of EL by regulating protease activity, as well as pathways involved in vascular ECM deposition.As highlighted earlier, LRP1 also regulates SMC phenotype, which affects SMC proliferation and migration.Recently, a pilot study conducted on human abdominal aortic aneurysm tissues implicated the association of LRP1 expression to the pathogenesis of aneurysm.119 This study reveals that LRP1 expression was significantly attenuated in abdominal aortic aneurysms tissues compared with normal aortic tissue.These studies are consistent with the genetic work in mice 19 and suggest that LRP1 may regulate aneurysm progression in humans as well.

SMC LRP1 Protects Against Excessive PDGF Signaling During Atherosclerosis and Restenosis
Under pathological conditions SMCs are induced to proliferate and migrate and contribute to the development of atherosclerosis and restenosis.Activation of the PDGF signaling pathway is associated with both of these events.2][123][124][125][126][127] The PDGF signaling pathway is activated when PDGF binds to the PDGF receptor (PDGFR)−β, which is a receptor tyrosine kinase that promotes cell growth and cell migration.Tight regulation of the PDGFR is critical, as excessive activation of the PDGF pathway induces tumor formation. 128,129One way by which this occurs is via LRP1, [16][17][18]77,78,114,130 which suppresses the PDGF signaling pathway by mechanisms that are not yet fully understood. This was elegntly demonstrated by generating smLRP1-mice on a background of LDLR deficiency.16 The studies found that smLRP1-/LDLR-mice were much more susceptible to cholesterol-induced atherosclerosis and displayed overexpression of the PDGFR along with excessive PDGFR signaling.The net effect was excessive proliferation of vascular SMCs resulting in a significantly enlarged aorta.In addition, smLRP1-mice displayed a disruption of the elastic layer and aneurysm formation.These effects could be inhibited by treatment of the mice with Gleevec, a known inhibitor of tyrosine kinases, including the PDGFRβ. Overall, the experiments reveal that LRP1 plays an important role in protecting the integrity of the vascular wall and reducing the extent of atherosclerosis by suppressing PDGFR activation.
How LRP1 regulates this pathway is not entirely clear at present.LRP1 is tyrosine phosphorylated on activation of the PDGFRβ at the terminal NPxY motif within the LRP1-ICD, which creates a docking site for phosphotyrosinebinding domain and Src homology 2 domain containing adaptor proteins. 77,79,130,1313][134][135] Shp-2 binds with high affinity to phosphorylated forms of the LRP1-ICD and its association with LRP1 attenuates PDGF-mediated signaling events. 131Another molecule that can associate with the LRP1-ICD is c-Cbl, a ubiquitin E3-ligase that regulates turnover of receptor tyrosine kinases, such as the PDGFRβ.Takayama et al 80 demonstrated increased turnover of the PDGFRβ in mouse embryonic fibroblasts lacking LRP1 when compared with fibroblasts expressing LRP1.These data suggest that LRP1 can regulate PDGFRβ turnover.However, these data do not explain the in vivo observations in smLRP1-mice, in which increased levels of active PDGFRβ and increased PDGF signaling are observed. 16

Macrophage LRP1 Is Also Atheroprotective
Lipid uptake by macrophage lipoprotein receptors and transporters have been implicated in atherogenesis by accelerating foam cell transformation. 136Interestingly, however, Hu et al 21 demonstrated that macrophage LRP1 has atheroprotective effects independent of its function in the uptake of lipoproteins.These studies revealed that macLRP1-mice on an apolipoprotein E/LDLR double knockout background resulted in an increase in total atherosclerotic lesion area and a higher frequency of advanced lesions.Furthermore, lesions from macLRP1-mice showed increased collagen content and a decrease in CD3 + T cells when compared with littermate controls.
In agreement with the atheroprotective role of macrophage LRP1, Overton et al 20 transplanted bone marrow from macLRP1-mice into lethally irradiated recipient mice deficient in the LDLR.After placing the mice on a high-fat diet, they demonstrated an increase in lipid deposition in the proximal aorta, as well as an increase in macrophage cellularity.Interestingly, no change in en face analysis of the lipid content of the aorta was noted.Macrophages from macLRP1-mice showed an increase in production of proinflammatory markers macrophage chemoattractant protein-1 and tumor necrosis factor-α. 137The ability of LRP1 to regulate the inflammatory response is suggested from studies demonstrating that LRP1 modulates the transition of macrophages from the classic M1 to alternative M2 phenotype. 138In addition, increased activity and synthesis of matrix metalloproteinase-9 in LRP1deficient macrophages correlated with a higher frequency of breaks in the EL.
Other studies have also confirmed the atheroprotective function of LRP1 by modulating the inflammatory response.Thioglycollate-elicited peritoneal macrophages from macLRP1-mice stimulated with lipopolysaccharide (LPS) showed increased secretion of tumor necrosis factor-α. 20ncreased production of tumor necrosis factor-α was also observed on LPS stimulation in peritoneal macrophages isolated from LDLR-deficient mice carrying an inactivating mutation in the LRP1-ICD NPxYxxL motif, 139 revealing that association of adaptor proteins with the LRP1-ICD is important for this activity. 76Insight into potential mechanism by which LRP1 can regulate the inflammatory response comes from the findings that LRP1 attenuates the transcription of LPS-inducible genes.LPS treatment of macrophages increases LRP1 shedding and γ-secretase-dependent release of the LRP1-ICD from the plasma membrane.The ICD is translocated to the nucleus where it limits LPS-activated transcription by promoting the nuclear export and subsequent degradation of interferon regulatory factor 3. 76 Inflammatory mediators, such as LPS and interferon-γ, have been shown to induce LRP1 shedding in cultured bone marrow macrophages isolated from C57BL/6 mice. 140Shed LRP1, consisting of the extracellular α-chain and a small portion of the transmembrane-containing β-chain, enhanced activation of p38 mitogen-activated protein kinases and upregulated transcription of macrophage chemoattractant protein-1, tumor necrosis factor-α, and interleukin-10 mRNA in RAW 264.7 mouse leukemic monocyte macrophage-like cell line macrophage-like cells.This study suggests that shed LRP1 generated during inflammation may modulate regulatory cytokine expression by macrophages; however, additional studies are needed to confirm the function of shed LRP1 in vivo.

LRP1 Modulates the Transforming Growth Factor-β Signaling Pathway
The effects of transforming growth factor (TGF)-β within the vasculature are complex.3][144][145][146] On vascular injury, TGFβ signaling also contributes to the repair process.The evidence for this activity of TGFβ includes gene transfer studies, 147 inhibition of TGFβ expression with ribozyme oligonucleotides, 148 or use of a recombinant soluble decoy TGFβ receptor II, 149 all of which substantiates that TGFβ contributes to vascular remodeling during restenosis.
Several studies support the notion that LRP1 is an important modulator of the TFGβ signaling pathway.Thus, Huang et al 81 demonstrated that 125I-labeled TGFβ could be crosslinked to LRP1, and that this interaction was inhibited by RAP, the LRP1 antagonist.These studies also demonstrated that murine fibroblasts in which LRP1 was genetically deleted were not sensitive to growth inhibition by TGFβ. 81The direct binding of both TGFβ1 and TGFβ2 to LRP1 was demonstrated by surface plasmon resonance experiments. 22Evidence is accumulating to suggest the in vivo importance of LRP1 in regulating the TGFβ signaling pathway.Thus smLRP1-mice maintained on an LDLR-deficient background displayed excessive nuclear accumulation of phosphorylated Smad2/3 in the vasculature 17 revealing activation of the TGFβ signaling pathway.In a carotid artery ligation model, macLRP1-mice demonstrated reduced intimal hyperplasia through modulation of the TGFβ signaling pathway. 22In these studies, quantitative reverse transcription polymerase chain reaction arrays revealed enhanced expression of TGFβ2, Pdgfa, and Eln in ligated vessels from macLRP1mice, whereas immunohistochemical analysis of carotid artery sections from macLRP1-mice confirmed enhanced expression of TGFβ2 during vascular remodeling and activation of the TGFβ signaling pathway by staining with antibodies specific to phosphorylated Smad2/3.Together, all of these studies confirm that LRP1 modulates the TGFβ signaling pathway, although the mechanism of how this occurs requires further study.

Perspective
It is clear that LRP1 is a key receptor in the regulation of plasma levels of fVIII.However, at present, key questions remain to be answered on the mechanism of how this could occur.Further, it remains to be established whether a mutant fVIII defective in binding to LRP1 would be a better product for the treatment of hemophilia A. Second, the in vivo significance of the TF pathway inhibitor-mediated clearance of fVII/TF complexes by LRP1 remains to be established.Finally, although several studies have documented clearly an important role for LRP1 in protecting the vasculature, our current understanding for this role is not yet complete.For example, recent findings suggest potential role for LRP1 in vascular development and in modulating appropriate formation of the extracellular matrix in the vasculature. 19The extensive EL disruption in the medial vessel wall of smLRP1-mice raises questions of whether this is entirely the result of excess protease activity or whether LRP1 contributes to the process of elastogenesis and vascular wall development.HtrA1 is the most recent addition to the extensive list of LRP1 ligands.Because HtrA1 is a key proteolytic enzyme leading to degradation of matrix proteins, deregulation of its levels in mice lacking LRP1 in the vessel wall suggests a role for this protease and also for LRP1, in aneurysm formation.This will need to be firmly established by additional genetic studies.
We can better understand the contribution of LRP1 in vessel wall homeostasis by first defining the role of this receptor in vascular SMC biology.Transmission electron microscopy analysis revealed that LRP1-deficient SMCs in adult vessels are in a more synthetic state rather than the expected fully differentiated contractile state.These data are supported by cell culture studies on aortic SMCs isolated from smLRP1mice, 18 and together, the results suggest a role for LRP1 in regulating SMC phenotype.This has to be further investigated to reveal the mechanisms by which LRP1 regulates SMC phenotypic alterations.Advances in our understanding of how LRP1 affects SMC adhesion and deadhesion processes and its role in the synthesis and modeling of the ECM are likely to give insight into the pathogenesis of vascular diseases such as Marfan syndrome and aortic aneurysms.
The well-studied LRP1-mediated uptake of growth factors and proteases that are generated in an inflammatory environment also requires a fresh examination to distinguish between endocytosis and removal of the growth factor versus a direct activation of signaling pathways on growth factor binding to LRP1.The variety and the number of the ligands that bind to LRP1 and to additional receptors known to initiated signaling pathways raises questions on the ability of LRP1 to mediate cross-talk between multiple signaling pathways and thereby fine tune responses to environmental cues.Finally, the mechanism by which LRP1 regulates the PDGF signaling pathway still needs to be firmly established.
Currently, there are no treatments to alter progression of vascular ECM diseases in patients.To date, studies suggest that smLRP1-mice represent an excellent model to examine the molecular events leading to vessel wall dysfunction.Additional studies defining the molecular mechanism by which LRP1 alters SMC phenotype in the vessel wall and regulates the integrity of the EL and matrix deposition are likely to identify potential therapeutic targets to maintain vessel wall function.

Figure 1 .
Figure 1.Canonical mode of ligand binding by low-density lipoprotein receptor-related protein-1 (LRP1).A, LRP1 is synthesized as a 600-kDa protein and is cleaved by furin into a light chain (arrow) that consists of an 85-kDa subunit containing the transmembrane and intracellular domain and a noncovalently 515-kDa amino-terminal fragment.The extracellular domain contains clusters (I, II, III, and IV) of LDL receptor type A repeats to which ligands bind.The cytoplasmic tail of the receptor contains 2 NPxY motifs (*).B, Structure of the receptor associated protein D3 domain lys256 LDL receptor complex showing the calcium ion stabilizing aspartic residues 147, 149, and 151.Tryptophan 144 forms Van der wall interactions with the K256 side groups (Protein Data Bank [PDB] accession number, 2FCW).C, Structure of the apoE receptor 2 reeling complex demonstrating a similar acidic pocket for lysine 2467 on reelin (PDB accession number, 3A7Q).

Figure 3 .
Figure 3. Genetic deletion of lrp1 from smooth muscle cells (SMCs) results in extensive disruptions in the vessel wall.Electron microscopy images of sibling control mice expressing low-density lipoprotein receptor-related protein-1 (LRP1; A and C) and smLRP1-mice (B and D) visualizing the intact (A) vs disrupted (B) lamella (*) including the internal elastic lamina (IEL).Compare higher magnification (C) of vascular SMCs in contact with organized extracellular matrix (ECM) with collagen fiber (col) in aorta of control mice with SMCs loosely in contact with unorganized collagen in aortas from smLRP1-mice (D).Medial SMCs in control aorta are contractile with classical hill-and-valley morphology (C).SMCs from smLRP1mice have prominent synthetic organelles (D, bracket).Proteolytic products of ECM in smLRP1-mice are cleared by phagocytosis by a macrophage (Mϕ; D; bar=2 µm for all images).Adapted with permission from Muratoglu et al.19 Authorization for this adaptation has been obtained both from the owner of the copyright in the original work and from the owner of copyright in the translation or adaptation.