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Lectin-like Oxidized Low-Density Lipoprotein (LDL) Receptor (LOX-1): A Chameleon Receptor for Oxidized LDL Bushra Zeya,† Albina Arjuman,‡ and Nimai Chand Chandra*,† †

Department of Biochemistry, All India Institute of Medical Sciences, Patna 801507, India Division of P&I, Indian Council of Medical Research, New Delhi 110 029, India

Biochemistry 2016.55:4437-4444. Downloaded from pubs.acs.org by DUQUESNE UNIV on 09/28/18. For personal use only.

‡

ABSTRACT: LOX-1, one of the main receptors for oxLDL, is found mainly on the surface of endothelial cells. It is a multifacet 52 kDa type II transmembrane protein that structurally belongs to the C-type lectin family. It exists with short intracellular N-terminal and long extracellular C-terminal hydrophilic domains separated by a hydrophobic domain of 26 amino acids. LOX-1 acts like a bifunctional receptor either showing pro-atherogenicity by activating the NFκB-mediated down signaling cascade for gene activation of pro-inflammatory molecules or playing an atheroprotective agent by receptormediated uptake of oxLDL in the presence of an anti-inflammatory molecule like IL-10. Mildly, moderately, and highly oxidized LDL show their characteristic features upon LOX-1 activation and its ligand binding indenture. The polymorphic LOX-1 genes are intensively associated with increased susceptibility to myocardial diseases. The splicing variant LOX IN dimerizes with the native form of LOX-1 and protects cells from damage by oxidized LDL. In the developing field of regenerating medicine, LOX-1 is a potential target for therapeutic intervention.

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be expressed majorly in vascular endothelial cells, placenta, and lung. Currently, it is thought that the detrimental effects of oxLDL in developing cardiovascular thrombosis could be aborted by inactivating LOX-1, which could be a target for generating a biotechnological tool in future therapy. There is a great body of evidence of oxidative stress in all stages of atherosclerosis; hence, oxLDL is now a popular target for exploring atherosclerotic stress. OxLDL acts via a variety of cell surface receptors such as SR-AI/AII, CD68/macrosialin, CD36, SR-BI/BII, etc. This led to the identification and characterization of a novel lectin-like receptor for oxidized LDL on the surface of endothelial cells by Sawamura and colleagues in 1997.12 Though Sawamura et al. had identified LOX-1 on the basis of the ability of endothelial cells to interact with oxidatively modified LDL through a pathway independent of the macrophage scavenger receptors, the functional role of LOX-1 in oxLDL internalization and trafficking remained elusive until the first report in this regard surfaced in 2008. Murphy et al.13 reported that LOX-1 trafficking was mediated via the dynamin-2 pathway studied in HeLa cells transfected with LOX-1 cDNA containing an engineered FLAG tag. Their site-directed mutagenesis studies also identified a tripeptide conserved motif (DDL) in a position proximal to the Nterminal end (cytosolic domain) of LOX-1 that is responsible for aiding this internalization.

therosclerosis is an age-linked slowly developing disease of the large- and medium-sized arteries. Atherogenesis is a sequence of events associated with the expression of adhesion molecules,1,2 recruitment of mononuclear cells to the endothelium,3 local activation of leukocytes followed by inflammation,4 lipid accumulation, and foam cell formation.5 The main predilection sites of manifestation of the atherosclerotic pathology are the deep intimal layers of large arteries such as the common carotid artery (at the bifurcation), the aorta (at the start of its branches), and the subclavian artery.6 Accumulation of low-density lipoproteins (LDL) in blood vessels, among many other lipids, is reported to be the major culprit in the generation of atherosclerotic plaque on the vessel wall. Resistance to clearance and longer persistence in blood vessels promote chemical oxidation of the existing LDL particles by dissolved oxygen and other oxidizing entities present in blood plasma. The resulting oxidized LDL (oxLDL) then turns out to be more and more pro-inflammatory by facilitating the formation of more oxLDL and other proinflammatory cytokines through vicious chain reactions.7 The vicious cycle is mediated through the interaction of oxLDL with its recently identified specific receptor called the lectin-like oxidized low-density lipoprotein receptor (LOX-1).8−10 Modified forms of lipoproteins can act directly on monocytes and macrophages. Both highly oxidized LDL (which is recognized by scavenger receptors but not by the classically known LDL receptor) and minimally modified LDL (which is still recognized by the LDL receptor) can increase the level of expression of certain macrophage scavenger receptors, resulting in a more effective clearance of oxLDL and enhancement of foam cell formation.11 LOX-1 is one such receptor and found to © 2016 American Chemical Society

Received: May 12, 2016 Revised: July 15, 2016 Published: July 15, 2016 4437

DOI: 10.1021/acs.biochem.6b00469 Biochemistry 2016, 55, 4437−4444

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Biochemistry Although many studies have demonstrated the possible mechanistic role of LOX-1 using hyperexpression model systems or knockout models, any “transient expression model” relating the in vivo physiological conditions has not yet been described, although the bifunctional character of LOX1 has been reported (Pro- and Anti-Inflammatory Response of LOX-1). Being bifunctional in nature, LOX-1 may act as a proand anti-atherogenic messenger. The central theme of this review is thus to justify LOX-1 as a central regulator in the atherogenic milieu.

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VARIANCE OF THE LOX-1 RECEPTOR Structure of the LOX-1 Protein. The lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) of Homo sapiens is a 52 kDa membrane-bound glycoprotein belonging to the Ctype lectin superfamily and consisting of 273 amino acid residues. It is mainly present on endothelial cells, macrophages, smooth muscle cells, and platelets.12 It is encoded by the OLR1 gene, which is a single-copy gene present in the p12.3−p13.2 region on chromosome 12.14 The LOX-1 gene is an inducible gene having TATA and CAAT boxes in the proximal part of the 5′ flanking region. The TATA box is located at −29 bp and the CAAT box at −99 bp.15 The LOX-1 gene is similar to the NK (Natural Killer) gene complex and like other NK cell receptors consists of four domains. These are the N-terminal cytoplasmic domain, the transmembrane domain, the NECK domain, and a C-terminal domain called a C-type lectin-like domain (CTLD). This CTLD has been experimentally confirmed as a ligand binding domain,15 and the NECK domain maintains the dimer structure. The human LOX-1 gene is 7000 bp long and consists of six exons and five introns. The size of exons 1−5 varies from 102 to 246 bp, and the sixth exon is longer (1722 bp).14 The 5′ untranslated region (5′UTR) and cytoplasmic domain are encoded by exon 1, while the remaining cytoplasmic and transmembrane domains are encoded by exon 2. The Neck region is encoded by the NECK domain or exon 3. Exons 4−6 encode the lectin- like domain and the 3′UTR.16 LOX-1 shows sequence homology with C-type lectins, which are proteins that recognize and bind to specific carbohydrate targets.8 The ligand binding domain of LOX-1 consists of three intramolecular disulfide bonds. Two of them are invariant disulfide bonds present in all the members of the C-type lectin-like domain, and the third one connects the first antiparallel β-sheets in the CTLD region to the linker part from the NECK segment as shown in Figure 1.17 In the case of human LOX-1, the disulfidelinked homodimer is present at C-140 on the cell surface.18 Alternative splicing of selective exons has shown three splice variants for LOX-1 transcripts. Transcript Variant 1. This variant is the full length mature LOX-1 that has all the exons. It is recruited to the plasma membrane and is functionally active in binding oxLDL and internalizing it mainly in the endothelial cells. Transcript Variant 2. This variant is a splice variant that lacks exon 4 and hence lacks a part of the ligand recognition domain. Transcript Variant 3. This variant is also a splice variant that lacks exon 5, the oxLDL binding region in the CTLD. This is an important variant that has been hypothesized to have a protective effect as against mature LOX-1. Thus far, Mango et al.19 are the only ones to have shown that this isoform of LOX1, termed LOXIN, has a protective effect in myocardial

Figure 1. Crystal structure of the H. sapiens mRNA sequence of LOX1. The intrachain disulfide bonds are shown as red balls and sticks.

infarction by forming “probable” nonfunctional dimers with mature LOX-1 in the ER and hence inhibiting its recruitment back to the membrane for further uptake of oxLDL. They have shown that macrophages of those subjects carry the “nonrisk” disease haplotype of the OLR1 gene. A relatively large amount of this particular variant decreases the cytotoxicity induced by oxLDL and hence could act as an anti-apoptotic component. The extracellular lectin-like domain of human LOX-1 is a heart-shaped homodimer with a central hydrophobic tunnel that extends through the entire molecule. The hydrophobic tunnel accommodates a cholesterol molecule, a fatty acid chain, and six or seven residues of the nonpolar peptide.20 LOX-1 identifies multiple ligands, including modified lipoprotein (such as oxLDL and acetylated LDL), polyanionic chemicals, anionic phospholipids, and cellular ligands. Thus, LOX-1 has versatile physiological functions. Species Variance of LOX-1 (rat and human). The amino acid sequence of human LOX-1 is similar to the rat LOX-1 amino acid sequence. According to a report by Nagase et al.,21 the rat LOX-1 amino acid sequence was almost 60% similar to the human LOX-1 amino acid sequence (Figure 2). Rat LOX-1 protein constitutes 364 amino acids, while the human protein forms contain 273 and 270 amino acids. Unlike human LOX-1, rat LOX-1 consists of triple repeats of a 46-amino acid motif between the transmembrane and lectinlike domain, which represents the NECK domain in human LOX-1. The repeats of the 46-amino acid domain made rat LOX-1 longer than its human counterparts.22 It is rich in glutamate, glutamine, leucine, and lysine residues. The 3′UTR of rat LOX-1 contains regions rich in A and U and polyadenylation signals. While both rat LOX-1 and human LOX-1 possess a singlecopy gene for LOX-1, the rat gene spans over 19 kb length and consists of eight exons; the human LOX-1 gene extends 7 kb 4438

DOI: 10.1021/acs.biochem.6b00469 Biochemistry 2016, 55, 4437−4444

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Biochemistry

leads to foam cells.26 In case of the human THP-1 cell line (macrophage type), exposure to mildly oxidized LDL led to accumulation of both cholesterol and cholesteryl ester in the lysosomal compartment.26 Moderately Oxidized LDL. Moderately oxidized LDL (15− 30 nmol of TBARS/mg of Apo B) contains a large amount of lipid hydoperoxide, which induces heme oxygenase-1 expression and enhances GSH levels to an extent greater than that seen with highly oxidized LDL.27 Moderately oxidized LDL also elevates the level of nuclear translocation of Nrf 2 and the level of phosphorylation of p38MAPK to a greater extent than other oxLDLs do. Highly Oxidized LDL. In highly oxidized LDL (>30 nmol of TBARS/mg of Apo B), LDL is modified to such an extent that it is not recognized by classical LDL receptors. These become ligands to another family of receptors called scavenger receptors. There is significant accumulation of free cholesterol because of the exposure of macrophages to LDL but little increase in the level of accumulation of cholesteryl esters. In a study by Roma et al., it was shown that there was almost 85% accumulation of free cellular cholesterol when the J774 macrophage cell line was incubated with extensively oxidized LDL but a very small increase in the level of cellular cholesteryl ester.28 In another similar study by two groups, Roma et al. and Brown et al. demonstrated that incubation of mouse peritoneal macrophages with extensively oxidized LDL resulted in levels of accumulation of free cholesterol increased approximately 40− 50% and only 5−10% accumulation of cholesteryl ester. Thus, the intensity of ester accumulation depends on the degree of oxidation of LDL.29,30 LOX-1 binds with more efficiently to a modified form of LDL such as oxidized LDL rather than untreated LDL, suggesting that LOX-1 recognizes modified Apo B. 31 Considering the degree of LDL oxidation, LOX-1 shows a higher affinity for moderately oxidized LDL than for extensively oxidized LDL.

Figure 2. Cellular orientation of human LOX-1 reported by Ohki et al.17 Reprinted from ref 17. Copyright 2005 Elsevier.

with only six exons. The difference in the properties of exons between rat and human LOX-1 is shown in Table 1. Table 1. Differences in the Functional Properties of Exons between the Rat and Human LOX-1 Genes position of the exon 1 2

3 4 and 5

6−8

rat LOX-1 encodes the 5′UTR and N-terminal 25 amino acids encodes the transmembrane domain

encodes 82 amino acids and repeat 1 in the extracellular domain encodes 46 amino acids and corresponds to repeat 2 (exon 4) and repeat 3 (exon 5) encodes 131 amino acids corresponding to the lectin-like domain and 3′UTR (exon 8)

human LOX-1 encodes the 5′UTR and cytoplasmic domain encodes the remainder of the cytoplasmic domain and transmembrane domain NECK domain encodes the lectin domain, including exon 6 encoding 3′UTR unknown

Susceptibility Variance of LOX-1 to Mildly, Moderately, and Highly Oxidized LDL. Expression of LOX-1 depends on the grade of oxLDL. In a study, it was reported that at 40 μg/mL mildly, moderately, and highly oxidized LDL, the levels of expression of LOX-1 were 26.1, 51.8, and 40.3%, respectively. At 80 μg/mL mildly, moderately, and fully oxidized LDL, they further increased to 40.3, 61.3, and 76.4%, respectively.23 Oxidation of LDL takes place in the subendothelial space of the arteries and not in circulation. Highly oxidized LDL possesses a very short half-life in plasma as it is cleared rapidly from the circulation. Sometimes small amounts of oxidized LDL are detected immunologically in normal plasma, and those amounts are increased in several diseases like diabetes and heart diseases.24 The oxidation of LDL leads to modification of lysine residues of the LDL protein. Almost 32% of lysines are modified in extensively oxidized LDL.25 Mildly Oxidized LDL. Mildly oxidized LDL (3−12 nmol of TBARS/mg of Apo B) is used to describe an oxLDL preparation that is used as a modified variant to be chemically distinguished from unmodified LDL, but they both can bind to LDL receptor. They are not identified by several scavenger receptors but have distinct biological activities that are not exhibited by unmodified LDL like induction of proinflammatory actions of endothelial cells and macrophages. In a study, it was shown that free cholesterol and cholesteryl ester were efficiently loaded in macrophages depending on species.26 In the case of mouse peritoneal macrophages, free cholesterol accumulated in the first 24 h of exposure and in the following hours almost 75% of free cholesterol was esterified and its excess stored as cytoplasmic cholesteryl ester droplets, which

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LOX-1 SIGNALING Role of NO and NFκB. NO, a short-lived gas, can freely diffuse through cells. The effect of nitric oxide can be propagated because of its interaction with thiol groups present on cysteine, glutathione, and heme proteins.32 It has been recognized as an important molecule in the regulation of apoptosis of cells and its viability. It has an extensive regulatory role in inflammatory response. NO readily reacts with molecular oxygen and superoxide radical, leading to oxidation of nitric oxide at physiological pH. This leads to formation of nitrite. NO also reacts with superoxide to form peroxynitrite, which is stable but can form nitrate and highly reactive OH radical and hence shows its proapoptotic and necrotic activity.33 Studies have shown that binding of oxLDL to LOX-1 increases the intracellular level of ROS such as superoxide anion (O2−) and hydrogen peroxide (H2O2). Intracellular nitric oxide reacts with superoxide anion, and there is, thus, a subsequent decrease in the NO level in cells. This condition leads to endothelial dysfunction. This is mainly due to the fact that oxLDL decreases endogenous superoxide dismutase activity, and increased nitric oxide synthase activity leads to an increased level of free radical formation and hypoxia. Nitric oxide synthase is an enzyme that is involved in the conversion of amino acid L-arginine into NO and L-citrulline and plays a crucial role in the regulation of vascular tone.34 4439

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Biochemistry

Figure 3. Mechanism of oxLDL signaling.

events that occur first in the chain of reaction of NFκB activation. Certain antioxidants inhibit activation of NFκB such as pyrrolidine, dithiocarbamate, and N-acetylcysteine.39 In addition, caffeic acid phenethyl ester (CAPE) is also a strong inhibitor of NFκB.40

Nitric oxide protects against vascular injury, inflammation, and thrombosis. It also inhibits adhesion of leukocytes to the endothelium and maintains the nonproliferative state of vascular smooth muscle cells and in turn limits platelet aggregation.35,36 Angiotensin II is a vasoconstrictor, inhibits NO action, and leads to production of ROS. NFκB, an oncogenic protein, regulates transcription of a variety of genes such as immune and inflammatory response genes (Figure 3). This transcription factor has a role to play in atherosclerosis. It is present as a heterodimer in the cytosol with NFκB1 (p50), Rel (p65), and p(56) subunits and bound to an inhibitor named IκB. NFκB, on activation, releases IκB and migrates from the cytosol to the nucleus of the cell and binds to specific DNA sequences and performs transcription.37 The transcribed genes encode pro-inflammatory and adhesion molecules such as TNF-α, ICAM-1, and VCAM-1. This NFκB is activated by inflammatory stimulation in macrophages, endothelial cells, smooth muscle cells, and T cells. These cells play an important role in atherosclerosis. A study by Maziere et al.38 has demonstrated that oxLDL stimulates NFκB in endothelial cells, smooth muscle cells, and fibroblasts and leads to cell injury. Thus, oxidative activation of NFκB in endothelial cells causes changes in cell phenotype and initiates atherosclerotic lesion formation. An intracellular ROS serves as a downstream messenger for various pathways leading to inactivation of NFκB.9 In a study, it was also demonstrated that oxidized LDL leads to activation of NFκB in bovine aortic endothelial cells (BAECs).9 The 5′ flanking region of the LOX-1 gene contains a consensus sequence of the NFκB binding site. This suggests that a certain inflammatory signal induces the LOX-1 gene and leads to activation of NFκB, causing transcriptional regulation.14 In certain reports, it has been mentioned that LOX-1 expression also depends on activation of NFκB induced by oxLDL due to generation of ROS. This thus produces the vicious cycle of oxLDL-induced LOX-1 signaling for the promotion of its proinflammatory activity. Furthermore, it has been reported that incubation of an antiLOX-1 monoclonal antibody inhibited NFκB activation induced by oxLDL. Thus, this suggests that binding of oxLDL to LOX-1 and subsequent formation of ROS are the

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PRO- AND ANTI-INFLAMMATORY RESPONSE OF LOX-1 LOX-1 is a bifunctional receptor with respect to the generation of a pro-inflammatory signal and faster utilization of ox-LDL in the presence of anti-inflammatory cytokines like IL-10.41 Vascular inflammation is transmitted by various cells that communicate among themselves through a chain of cytokine receptors and their cytokine mediators. Cytokine shows autocrine, paracrine, and juxtacrine signaling properties. Attachment of cytokine to its receptor initiates a series of intracellular signals, including activation of kinases and transcription factors.42 An increased level of expression of LOX-1 in the intima of an atherosclerotic lesion is also due to the presence of inflammatory cytokines.43 Expression of various adhesion molecules such as VCAM-1 and ICAM-1 is induced by pro-inflammatory cytokines such as IL-1β and TNFα. The expressions of these cytokines are induced by the interaction of LOX-1 with oxLDL and CD40/ CD40L ligand interaction.44 Large numbers of cytokines such as IL-1β, IL-6, IL-8, IL-12, IL-18, IFNγ, and TNFα act as pro-inflammatory markers. These pro-inflammatory cytokines are also pro-atherogenic. IL1β acts through the p38MAPK signaling pathway and elicits expression of cytokines and adhesion molecules. IL-6 is synthesized by endothelial cells, vascular smooth muscle cells, and macrophages. The level of expression of IL-6 increases in patients with unstable angina and coronary artery disease.45 It functions through the JAK/STAT family of signal transducers. IL-6 enhances the expression of CAM (cell adhesion molecule) on endothelial cells. It also functions as a contributing factor in the extravasations of leukocytes in the atherosclerotic lesions. These cytokines are inter-related in their expressions, and expression of TNFα is a common link in most of the cases. TNFα is a pro-inflammatory cytokine and functions through 4440

DOI: 10.1021/acs.biochem.6b00469 Biochemistry 2016, 55, 4437−4444

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Biochemistry

Studies suggest an inverse relation between the level of LOXIN expression and the incidence of myocardial infarction in humans.19 Thus, the pro-apoptotic effect of LOX-1 can be barred by co-expression of LOXIN in a dose-dependent manner. Another study also suggests the hetero-oligomerization of the naturally occurring isoforms of LOX-1. LOXIN and LOX-1 in joint adherence result in the disruption of the functional properties of LOX-1 and increase the resistance to oxLDL-induced apoptosis.57 Thus, these findings make LOXIN a new target for the treatment of atherosclerosis. Another variant of LOX-1 that shows a G to C transition at position 501 leading to Lys-to-Asn conversion at position 167 of the peptide has been observed. It is located in the C-type lectin-like domain. The conversion of Lys to Asn leads to a decreased level of binding and internalization of oxLDL. Thus, it can be suggested that the existence of the LOX-1 gene variant plays an important role in the onset of atherogenesis.58

the p38/MAPK and NFκB signaling pathway, which is again linked to LOX-1 expression. In a study, it was demonstrated that there was an increased level of expression of LOX-1 and SR-A (scavenger receptor-A) mRNA in the presence of TNFα and IL-6.46 There have also been reports that expression of IL-6 is influenced by TNFα in THP-1 cells.46 In another report, it was proven that production of proinflammatory cytokines, such as TNFα and IL-1, induces LOX1 expression that in turn activates the NFκB signaling pathway.47 Increased production of IL-8 and LOX-1 expression was also observed in the THP-1 cell line with an increased level of oxLDL.48 Mattaliano et al. in their work have identified ROCK2 (Rho-associated, coiled-coil-containing protein kinase 2), a kinase acting as LOX-1 associating molecule. Accumulation of oxLDL stimulates ROCK2 to produce IL8.49 IL-1α, IL-1β, and TNFα have been reported to upregulate LOX-1 expression in cultured smooth muscle cells.50 Interdependency of LOX-1 and TNFα has been reported in one of the studies also. It was demonstrated that on one hand TNFα induces TNFR1 (receptor of TNFα) to generate LOX-1 and on the other hand shows an increased level of extracellular accumulation of oxLDL.51 TGFβ signals through smad3 and smad4 by interacting with 12-O-tetradecanoyl-13-acetate responsive elements (TREs) as well as AP-1 and activates TGFβ-dependent gene transcription. The consensus nucleotide sequence corresponding to TRE is present in the 5′ flanking region of the LOX-1 gene. Therefore, the smad-TRE pathway is responsible for LOX-1 expression.52 In a study, it is shown that TGFβ induces LOX-1 expression in cultured vascular endothelial cells, smooth muscle cells, and macrophages.53 Expression of IL-10 leads to a decreased level of cell damage and apoptosis in atherosclerotic plaques, and thus, IL-10 is regarded as an atheroprotective agent in nature.54 It exploits various mechanisms such as attenuation of inflammatory gene expression in many cell types, inhibition of T cell proliferation, and inhibition of antigen presentation. IL-10 activates the JAK/ STAT pathway, mostly STAT 3. It can even inhibit TNFαinduced MAPK signaling and NFκB activation in monocytes, macrophages, endothelial cells, and VSMC.55 IL-10 reduces the atherogenic propensity due to clearance of the oxLDL particle via LOX-1-mediated cellular uptake and acts as an atheroprotectant.41 Almost 60% reduced fatty lesion was observed in mice, which was electro-transferred with IL-10 cDNA. Thus, IL-10 makes LOX-1 anti- atherogenic.55

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LOX-1: A POTENTIAL TARGET IN CARDIOVASCULAR THERAPY Oxidized LDL plays a pathological role in the proliferation and development of atherosclerosis. Several therapeutic strategies have been developed, which reduces the plasma oxLDL levels of compounds such as naturally occurring antioxidants and antihypertensive agents.59 These agents either inhibit oxLDL formation or remove oxLDL from circulation, thus preventing atherogenesis.60 Several antioxidants such as tanshionone IIA,61 curcumin,62 berberine,63 and resveratrol64 prevent atherosclerosis by inhibiting the generation of oxidized LDL. They also inhibit expression of LOX-1 by inactivating the LOX1 signaling pathway as a result of the reduced circulating oxLDL level. Certain antihypertensive agents such as calcium channel blockers (CCB) and AT1R blockers (ARB) also limit and decrease the incidence of atherosclerosis and other cardiovascular events.65 Nifedine, a calcium channel blocker, has an inhibiting effect on LOX-1. It prevents the apoptosis of endothelial cells by downregulation of LOX-1. Various other studies demonstrate that the application of LOX-1 antibodies,66 antisense RNA,67 and miRNA68,69 also block LOX-1, thus laying a foundation for the development of therapeutic strategies. Our laboratory is also in the process of developing LOX-1 specific siRNAs70 with a target for generating biotechnology-based therapy in next-generation medicine. A mouse monoclonal antibody has also been developed, which inhibits LOX-1 activation and binding of ligands to LOX-1.71 It has been demonstrated in vitro that abJTX92 prevents the binding and internalization of oxLDL to LOX-1 in human artery endothelial cells.66 The LOX-1 antibody also inhibits the expression of adhesion molecules and eNOS. In the case of adult mouse cardiomyocytes, it was observed that anti-LOX-1 antibodies diminish the level of AngII-mediated oxidative stress and the level of expression of NADPH oxidase and NFκB,62 thus reducing the level of ROS and in turn preventing atherosclerosis. In another study by Cao et al.,72 it was demonstrated that a polyclonal antibody against Fc, cross-linked via LOX-1 Fc fusion protein, inhibited the binding of oxLDL to LOX-1. Recently in a study, it has been reported that intraperitoneal administration of the anti-LOX-1 antibody in rats reduces the level of activation and expression of LOX-1 and was found to be a novel therapeutic target for neonatal hypoxic-ischemic encephalopathy (HIE).73 The human LOX-1 monoclonal antibody has also been developed

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VARIANTS IN LOX-1 AND ITS ATHEROGENIC SENSITIVITY The OLR1 gene encodes LOX-1 receptors. It is mapped in human chromosome 12p, 12.3−13.1. Various studies have shown certain genetic variation in the OLR1 gene, and these variants are associated with coronary artery disease.19 Singlenucleotide polymorphisms (SNPs) have been identified in LOX-1 at intron 4 (G → A), intron 5 (T → G), and 3′UTR (T → C).56 The SNPs give rise to a splicing variant LOXIN, lacking exon 5. It is deficient in the ligand binding domain of LOX-1. The size of LOXIN protein has been predicted to be 21.4 kDa.57 The polypeptide consists of 188 amino acids, the N-terminus of wild-type LOX-1, and an altered amino acid at its C-terminus. LOXIN dimerizes with the native form of LOX1 and protects cells from damage by oxidized LDL. This reduces the level of expression of LOX-1 on the plasma membrane and the level of binding of oxidized LDL to LOX-1. 4441

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Biochemistry using Xenomouse. The antibody prevents oxLDL-induced ROS formation, RhoA/Rac1 activation, and MCP-1 expression.69 Antisense technology has also proven to be an effective strategy for the suppression of atherosclerosis and other cardiac diseases. Antisense oligonucleotides have been developed to lower the LOX-1 level. It was recently reported by Takedatsu et al. that schizophyllan (SPG) can be used as a delivery system for oligonucleotides (ODNs). Schizophyllan is a polysaccharide that belongs to the β(1−3)-glucan family.67 This delivery system is advantageous as it is stable in the in vivo system and not a substrate for deoxyribonuclease. It is internalized easily and efficiently by macrophages through the lectin-1 receptor. Amati et al. used this system to administer antisense olr1, i.e., SPG/olr1AS, in ApoE knockout mice and found that there was significant downregulation of LOX-1 mRNA and protein in the aorta of mice. An almost 63% reduction in the LOX-1 protein level was observed in the treated mice.74 miRNA and/or siRNA can also be a target for an effective therapeutic strategy. miRNAs are small endogenous noncoding RNAs approximately 21 nucleotides in length. They regulate the expression of various protein-coding genes post-transcriptionally. In a study, it has been demonstrated that there is a binding site for miRNA let-7g in the 3′UTR of LOX-1 mRNA. Transfection of let-7g resulted in the inhibition of oxLDLinduced expression of LOX-1.75 In another study by Ding et al.,68 they reported that the miRNA let-7g blocked LOX-1 expression as well as uptake of oxLDL in human aortic smooth muscle cells (HASMCs). Small interfering RNA (siLOX-1) also prevents oxLDL-induced activation of Rho A (Ras homologue gene family, member A) and Rac 1 (Ras-related C3 botulinum toxin substrate 1).69 siRNA for LOX-1 was also found to partially inhibit CRP (C-reactive protein) binding.76 In a study, it was reported that transfection of bovine aortic endothelial cells with siLOX-1 prevented the expression and upregulation of LOX-1.77 Certain other small molecules such as procyanide, a polyphenol compound present in red wine and apples, also prevent binding of oxLDL to LOX-1.78 In the recent study, Thakkar et al.79 showed that a virtual screening technique can also be used to identify the LOX-1 inhibitor. Two of the lead molecules, Mol-4 and Mol-5, were seen to strongly bind with LOX-1 and inhibit the uptake of oxLDL. Another study has also revealed that pretreatment with metformin reduces the level of oxLDL-induced endothelial apoptosis by increasing the level of expression of SIRT1.80 Thus, all these probes, such as antibodies, antisense oligonucleotides, siRNA, and miRNA, are the fast-emerging tools of biotechnology that can be used for therapeutic trials for developing the target missile for treating atherosclerosis. LOX-1 is now a novel therapeutic target for the treatment of cardiovascular diseases.

in-depth knowledge of LOX-1 signaling over pro-inflammatory gene transcription.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: chandra_nc1@rediffmail.com. Notes

The authors declare no competing financial interest.

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REFERENCES

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FUTURE PROSPECTS LOX-1 is one of the major mediators of the genesis of atherosclerosis. Expression of LOX-1 is regulated by pro- and anti-inflammatory cytokines. Therapeutic intervention in its signaling pathway may help as a preventive measure in atherosclerotic lesions. Gene therapy by post-transcriptional regulation of the receptor protein, e.g., modulation by siRNA, miRNA, shRNA, etc., may also be advantageous in lowering the risk of atherosclerosis and its related disorders. Thus, LOX-1 may be a prospective therapeutic target in combating atherosclerosis via the exploration of the understanding and 4442

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Biochemistry

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