Generation and characterization of chicken monoclonal antibodies against human LOX-1

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is the major receptor for oxidized LDL (oxLDL), and plays a key role in the pathogenesis of atherosclerosis and cardiovascular diseases. Monoclonal antibodies (mAbs) specific for human LOX-1 (hLOX-1) were generated by a phage display technique using chickens immunized with recombinant hLOX-1 (rhLOX-1). A total of 53 independent scFv clones reactive for rhLOX-1 were obtained. Of the 53 clones, 49 recognized the C-type lectin-like domain (CTL domain), which contributes to the binding of oxLDL. Of these, 45 clones inhibited oxLDL-binding with LOX-1. Furthermore, some of these clones cross-reacted with rabbit, pig and/or mouse LOX-1. For possible application as therapeutic agents in the future, two cross-reactive mAbs were re-constructed as chicken-human chimeric antibodies. The chimeric antibodies showed similar characteristics compared to the original antibodies, and inhibited oxLDL binding to LOX-1 expressed on CHO cells. The results obtained in this study indicate that anti-LOX-1 mAbs might be useful tools for functional analyses and development of therapeutic agents for cardiovascular indications such as atherosclerosis.     

Cloning and expression of human lectin-like oxidized low density lipoprotein receptor-1 in<Emphasis Type="Italic"> Pichia pastoris</Emphasis>

Lectin-like oxidatively-modified LDL receptor-1 (LOX-1) is a major receptor for oxidized low-density lipoprotein (oxLDL) in aortic endothelial cells. Human LOX-1 (hLOX-1) gene (cDNA) was cloned from the monocytic leukemic cell line THP-1 and expressed in Pichia pastoris. The recombinant protein (rhLOX-1) was purified by his-tag affinity chromatography. Preliminary identification was performed by Western blot analysis and a ligand-receptor binding assay showed that the protein had specific oxLDL-binding activity.Revisions requested 21 September 2004; Revisions received 10 November 2004     

Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds

The lectin-like oxidized low-density lipoprotein scavenger receptor (LOX-1) is a pro-inflammatory marker and Type II membrane protein expressed on vascular cells and tissues. The LOX-1 extracellular domain mediates recognition of oxidized low-density lipoprotein (oxLDL) particles that are implicated in the development of atherosclerotic plaques. To study the molecular basis for LOX-1-mediated ligand recognition, we have expressed, purified and refolded a recombinant LOX-1 protein and assayed for its biological activity using a novel fluorescence-based assay to monitor binding to lipid particles. Overexpression of a hexahistidine-tagged cysteine-rich LOX-1 extracellular domain in bacteria leads to the formation of aggregates that accumulated in bacterial inclusion bodies. The hexahistidine-tagged LOX-1 molecule was purified by affinity chromatography from solubilized inclusion bodies. A sequential dialysis procedure was used to refold the purified but inactive and denatured LOX-1 protein into a functionally active form that mediated recognition of oxLDL particles. This approach allowed slow LOX-1 refolding and assembly of correct intrachain disulfide bonds. Circular dichroism analysis of the refolded LOX-1 molecule demonstrated a folded state with substantial alpha-helical content. Using immobilized recombinant, refolded LOX-1 we demonstrated a 70-fold preferential recognition for oxLDL over native LDL particles. Thus, a protein domain containing intrachain disulfide bonds can be reconstituted into a functionally active state using a relatively simple dialysis-based technique.     

Low-density lipoproteins induce the renin-angiotensin system and their receptors in human endothelial cells.

Increased levels of low-density lipoproteins are well-established risk factors of endothelial dysfunction and the metabolic syndrome. In this study, we evaluated the effect of native low-density lipoprotein (nLDL) and oxidized LDL (oxLDL) on the expression of genes of the renin-angiotensin system (angiotensin-converting enzyme, ACE; angiotensin II type 1 receptor, AT(1)) and their receptors (low-density lipoprotein receptor: LDLR; lectin-like oxLDL receptor: LOX-1; toll-like receptor 4: TLR4) in primary cultures of human umbilical vein endothelial cells. ACE and AT(1) expressions were significantly increased after stimulation with nLDL and oxLDL. OxLDL receptor LOX-1 showed a maximum induction after 7 hours. Increased LOX-1 protein expression in response to oxLDL could be blocked by a LOX-1-specific antibody. TLR4 expression was increased by nLDL and oxLDL as well. We conclude that LDL and oxLDL can activate the renin-angiotensin system and their receptors LDLR, LOX-1, and TLR4 in human endothelial cells. These data suggest a novel link between hypercholesterolemia and hypertension in patients with the metabolic syndrome.     

The expression of recombinant human LOX-1 and identifying its mimic ligands by fluorescence polarization-based high throughput screening

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was identified as a major receptor for oxidized low-density lipoprotein (oxLDL) in endothelial cells. LOX-1 critically mediates the endothelial dysfunction and the progression of atherosclerosis by oxLDL stimulation. It might be an important target for vascular endothelium. In order to obtain human LOX-1 and identify its mimic ligand for facilitating the study of LOX-1 function, a recombinant plasmid pPIC9K-His-hLOX-1 was structured and expressed human LOX-1 in Pichia pastoris GS115. Western blot analysis ensured the expressed recombinant human LOX-1 protein and a receptor-ligand binding assay showed that it had high binding affinity with oxLDL. With this receptor protein, a competitive fluorescence polarization-based high throughput screening method was established in a 384-well microplate to isolate the mimic ligands of human LOX-1. The evaluating parameter Z' value of 0.72 for this method showed that fluorescence polarization-based high throughput screening assay was robust and the results had a high reliability. By the fluorescence polarization-based high throughput screening assay, a total of 20,316 chemicals were screened, and 2 chemicals were identified that they have a high affinity with human LOX-1. Competitive uptake DiI-oxLDL assay by human LOX-1 transfected CHO-K1 cells further confirmed that two chemicals block the uptake of DiI-oxLDL. And the preliminary results indicated that isolated mimic ligands may act as a function of antagonist. The discovery of human LOX-1 mimic ligand would benefit to further study the function of LOX-1 and identify a novel avenue for prevention and treatment atherosclerosis.     

Induction of the oxLDL receptor LOX-1 by endothelin-1 in human endothelial cells.

In this study, we analyzed the effect of endothelin-1 (ET-1) on expression of the lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 LOX-1 and on oxLDL uptake in primary cultures of human umbilical vein endothelial cells (HUVEC). LOX-1 mRNA was quantified by standard-calibrated competitive RT-PCR, LOX-1 protein expression by Western analysis and endothelial oxLDL uptake using DiI-labeled oxLDL. ET-1 induces LOX-1 mRNA expression, reaching its maximum after 1 h (160 +/- 14% of control, 100 nM ET-1, P < 0.05). This increased ET-1-mediated LOX-1 mRNA expression could be inhibited by endothelin receptor B antagonist BQ-788. In addition, ET-1 stimulates LOX-1 protein expression and oxLDL uptake in HUVEC. The augmented oxLDL uptake by ET-1 is mediated by endothelin receptor B, but not by protein kinases. These data support a new pathophysiological mechanism how locally and systemically increased ET-1 levels could promote LOX-1-mediated oxLDL uptake in human endothelial cells and the development and progression of endothelial dysfunction and atherosclerosis.     

Refolding and characterization of the functional ligand-binding domain of human lectin-like oxidized LDL receptor

Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a type II membrane protein that can recognize a variety of structurally unrelated macromolecules, plays an important role in host defense and is implicated in atherogenesis. To understand the interaction between human LOX-1 and its ligands, in this study the functional C-type lectin-like domain (CTLD) of LOX-1 was reconstituted at high efficiency from inactive aggregates in Escherichia coli using a refolding technique based on an artificial chaperone. The CD spectra of the purified domain suggested that the domain has alpha-helical structure and the blue shift of Trp residues was observed on refolding of the domain. Like wild-type hLOX-1, the refolded CTLD domain was able to bind modified LDL. Thus, even though CTLD contains six Cys residues that form disulfide bonds, it recovered its specific binding ability on refolding. This suggests that the correct disulfide bonds in CTLD were formed by the artificial chaperone technique. Although the domain lacked N-glycosylation, it showed high affinity for its ligand in surface plasmon resonance experiments. Thus, unglycosylated CTLD is sufficient for binding modified LDL.     

No upregulation of lectin-like oxidized low-density lipoprotein receptor-1 in serum-deprived EA.hy926 endothelial cells under oxLDL exposure, but increase in autophagy

The oxidized low-density lipoprotein (oxLDL)-dependent activation of the lectin-like oxLDL receptor-1 (LOX-1) triggers apoptosis in vascular cells and appears to be involved in atherosclerosis. Autophagy might be an alternate to apoptosis in endothelial cells. The EA.hy926 endothelial cell line has been reported to undergo necrosis under oxLDL stimulation. For this reason, we studied the expression of LOX-1 and its oxLDL-dependent function in EA.hy926 cells under serum starvation. Untreated and oxLDL-treated cells expressed the LOX-1 protein at similar levels 6h after starvation. After 24h without oxLDL and with native LDL (nLDL), statistically significant higher levels were found in LOX-1 than in the oxLDL-treated probes. The oxLDL cultures with low LOX-1 expression displayed stronger features of autophagy than those with nLDL as there were remodelling of actin filaments, disrupture of adherens junctions (immunofluorescence staining), and autophagosomes with the characteristic double membrane at the ultrastructural level. For the advanced oxLDL exposure times (18 and 24 h), autophagic vacuoles/autophagolysosomes were morphologically identified accompanied by a decrease in lysosomes. The autophagosome marker protein MAP LC3-II (Western blotting) was significantly augmented 6 and 18 h after oxLDL treatment compared with cultures treated with nLDL and medium alone. Signs of apoptosis were undetectable in cultures under oxLDL exposure, yet present under staurosporin (apoptosis inducer), i.e. presence of apoptotic bodies and cleaved caspase 3. We conclude that serum starvation upregulates LOX-1 in EA.hy926 cells, whereas the additional oxLDL treatment downregulates the receptor and intensifies autophagy probably by increase in oxidative stress.     

Activation-dependent surface expression of LOX-1 in human platelets

Lectin-like oxidized LDL receptor-1 (LOX-1) was initially identified as an oxidized LDL receptor in aortic endothelial cells. Here we identified LOX-1 mRNA and protein in human platelets in addition to recent findings on the expression in macrophages and smooth muscle cells. The presence of LOX-1 was further confirmed in the megakaryocytic cell lines. Flow cytometric analyses revealed that LOX-1 was exposed on the surface of platelets in an activation-dependent manner. Consistently, the activation-dependent binding of OxLDL to platelets was mostly inhibited by anti-LOX-1 antibody. Immunohistochemistry of the atherosclerotic plaque from a patient with unstable angina pectoris (UAP) revealed accumulation of LOX-1 protein at the site of thrombus. As LOX-1 recognizes and binds activated platelets, exposure of LOX-1 on activated platelets surface might assist thrombosis formation.     

The essential role of p38 MAPK in mediating the interplay of oxLDL and IL-10 in regulating endothelial cell apoptosis

Interleukin-10 (IL-10) may have therapeutic potential in various inflammatory diseases, including atherosclerosis, as it can inhibit oxLDL-induced foam cell formation and apoptosis in macrophages. This study investigated the effect of IL-10 on mitogen-activated protein kinase (MAPK) activation, and apoptosis induced by oxidized low-density lipoprotein (oxLDL) in cultured human umbilical vein endothelial cells (HUVEC). The results demonstrated that IL-10 significantly blocked the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) and apoptosis induced by oxLDL. The inhibitory effect of IL-10 on oxLDL-induced apoptosis was partially dependent on reduced p38, but not JNK, phosphorylation. This study also discovered a linkage between IL-10 and p38 MAPK signaling in oxLDL-induced endothelial cell apoptosis. Interestingly, this study found that lectin-like oxidized LDL receptor-1 (LOX-1) was the only scavenger receptor, on the surface of HUVEC, that was upregulated by oxLDL and the increase in LOX-1 was not suppressed by IL-10. This study confirmed that IL-10 significantly upregulated the expression of suppressor of cytokine signaling-3 (SOCS3), whereas SOCS3 knockdown by siRNA effectively blocked the inhibitory effect of IL-10 on p38 MAPK-dependent apoptosis induced by oxLDL. These results showed for the first time, that IL-10 modulated oxLDL-induced apoptosis by upregulating SOCS3, which then interrupted p38 MAPK activation in endothelial cells. These findings support the essential role of p38 MAPK in the interplay of oxLDL and IL-10 in endothelial apoptosis.     

Roles of lectin-like oxidized LDL receptor-1 and its soluble forms in atherogenesis

Lectin-like oxidized LDL receptor (LOX)-1 is a type II membrane protein that belongs to the C-type lectin family of molecules, which can act as a cell-surface endocytosis receptor for atherogenic oxidized LDL. LOX-1 can support binding, internalization and proteolytic degradation of oxidized LDL, but not of significant amounts of acetylated LDL, which is a well-known high-affinity ligand for class A scavenger receptors and scavenger receptor expressed by endothelial cells (SR-EC). LOX-1 is initially synthesized as a 40-kDa precursor protein with N-linked high mannose-type carbohydrate, which is further glycosylated and processed into a 50-kDa mature form. LOX-1 expression is not constitutive, but can be induced by proinflammatory stimuli, such as tumour necrosis factor-alpha, transforming growth factor-beta and bacterial endotoxin, as well as angiotensin II, oxidized LDL itself and fluid shear stress. In addition, LOX-1 expression is detectable in cultured macrophages and activated vascular smooth muscle cells. In vivo, endothelial cells that cover early atherosclerotic lesions, and intimal macrophages and smooth muscle cells in advanced atherosclerotic plaques can express LOX-1. Cell-surface LOX-1 can be cleaved through some protease activities that are associated with the plasma membrane, and released into the culture media. Purification of soluble LOX-1 and the N-terminal amino-acid sequencing identified the two cleavage sites (Arg86-Ser87 and Lys89-Ser90), both of which are located in the membrane proximal extracellular domain of LOX-1. Measurement of soluble LOX-1 in vivo may provide a novel diagnostic tool for the evaluation and prediction of atherosclerosis and vascular disease.     

Pterostilbene protects vascular endothelial cells against oxidized low-density lipoprotein-induced apoptosis in vitro and in vivo

Vascular endothelial cell (VEC) apoptosis is the main event occurring during the development of atherosclerosis. Pterostilbene (PT), a natural dimethylated analog of resveratrol, has been the subject of intense research in cancer and inflammation. However, the protective effects of PT against oxidized low-density lipoprotein (oxLDL)-induced apoptosis in VECs have not been clarified. We investigated the anti-apoptotic effects of PT in vitro and in vivo in mice. PT at 0.1–5 μM possessed antioxidant properties comparable to that of trolox in a cell-free system. Exposure of human umbilical vein VECs (HUVECs) to oxLDL (200 μg/ml) induced cell shrinkage, chromatin condensation, nuclear fragmentation, and cell apoptosis, but PT protected against such injuries. In addition, PT injection strongly decreased the number of TUNEL-positive cells in the endothelium of atherosclerotic plaque from apoE^−/− mice. OxLDL increased reactive oxygen species (ROS) levels, NF-κB activation, p53 accumulation, apoptotic protein levels and caspases-9 and -3 activities and decreased mitochondrial membrane potential (MMP) and cytochrome c release in HUVECs. These alterations were attenuated by pretreatment with PT. PT inhibited the expression of lectin-like oxLDL receptor-1 (LOX-1) expression in vitro and in vivo. Cotreatment with PT and siRNA of LOX-1 synergistically reduced oxLDL-induced apoptosis in HUVECs. Overexpression of LOX-1 attenuated the protection by PT and suppressed the effects of PT on oxLDL-induced oxidative stress. PT may protect HUVECs against oxLDL-induced apoptosis by downregulating LOX-1-mediated activation through a pathway involving oxidative stress, p53, mitochondria, cytochrome c and caspase protease. PT might be a potential natural anti-apoptotic agent for the treatment of atherosclerosis.     

Chaperonin-containing TCP-1 complex directly binds to the cytoplasmic domain of the LOX-1 receptor

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a scavenger receptor that binds oxidized low-density lipoprotein (OxLDL) and has a role in atherosclerosis development. The N-terminus intracellular region (cytoplasmic domain) of LOX-1 mediates receptor internalization and trafficking, potentially through intracellular protein interactions. Using affinity isolation, we identified 6 of the 8 components of the chaperonin-containing TCP-1 (CCT) complex bound to LOX-1 cytoplasmic domain, which we verified by coimmunoprecipitation and immunostaining in human umbilical vein endothelial cells. We found that the interaction between CCT and LOX-1 is direct and ATP-dependent and that OxLDL suppressed this interaction. Understanding the association between LOX-1 and the CCT complex may facilitate the design of novel therapies for cardiovascular disease.     

Unsaturated FAs prevent palmitate-induced LOX-1 induction via inhibition of ER stress in macrophages

Palmitic acid (PA) upregulates oxidized LDL receptor-1 (LOX-1), a scavenger receptor responsible for uptake of oxidized LDL (oxLDL), and enhances oxLDL uptake in macrophages. However, the precise underlying mechanism remains to be elucidated. PA is known to induce endoplasmic reticulum (ER) stress in various cell types. Therefore, we investigated whether ER stress is involved in PA-induced LOX-1 upregulation. PA induced ER stress, as determined by phosphorylation of PERK, eIF2α, and JNK, as well as induction of CHOP in macrophage-like THP-1 cells. Inhibitors [4-phenylbutyric acid (PBA), sodium tauroursodeoxycholate (TUDCA), and salubrinal] and small interfering RNA (siRNA) for the ER stress response decreased PA-induced LOX-1 upregulation. Thapsigargin, an ER stress inducer, upregulated LOX-1, which was decreased by PBA and TUDCA. We next examined whether unsaturated FAs could counteract the effect of PA. Both oleic acid (OA) and linoleic acid (LA) suppressed PA-induced LOX-1. Activation of the ER stress response observed in the PA-treated cells was markedly attenuated when the cells were cotreated with OA or LA. In addition, OA and LA suppressed thapsigargin-induced LOX-1 upregulation with reduced activation of ER stress markers. Our results indicate that activation of ER stress is involved in PA-induced LOX-1 upregulation in macrophages, and that OA and LA inhibit LOX-1 induction through suppression of ER stress.     

Lectin-like oxidized LDL receptor-1 as extracellular chaperone receptor: its versatile functions and human diseases

Well-known coronary risk factors such as hyperlipidemia, hypertension, smoking, and diabetes are reported to induce the oxidative stress. Under the oxidative stress, low-density lipoprotein (LDL) is oxidatively modified in the vasculature, and formed oxidized LDL induces endothelial dysfunction, expression of adhesion molecules and apoptosis of vascular smooth muscle cells. It has become evident that these cellular responses induced by oxidized LDL are mediated by lectin-like oxidized LDL receptor-1 (LOX-1). LOX-1 was originally identified from cultured aortic endothelial cells as a receptor for oxidized LDL; however, recent investigations revealed that LOX-1 has diverse roles in the host-defense system and inflammatory responses, and it is involved in the pathogenesis of various diseases such as atherosclerosis-based cardiovascular diseases and septic shock. Beside oxidized LDL, LOX-1 recognizes multiple ligands including apoptotic cells, platelets, advanced glycation end products, bacteria, and heat shock proteins (HSPs). The HSPs function as a chaperone to affect protein folding of newly synthesized or denatured proteins. There are accumulating evidences that the HSPs released into the extracellular space have potent biological activities and it may work as a kind of cytokines. It is demonstrated that LOX-1 works as a receptor for HSP70, since it has high affinity for HSP70. The interaction of LOX-1 with HSP70 is involved in the cross-presentation of antigen. Given the potent and wide variety of biological activities, more understanding their interaction provides potential therapeutic strategy for various human diseases.     

Lipoprotein-X reduces LDL atherogenicity in primary biliary cirrhosis by preventing LDL oxidation

Hypercholesterolemic human LDL contains oxidized subfractions that have atherogenic properties. Paradoxically, atherosclerosis incidence is low in patients with primary biliary cirrhosis (PBC), a disease characterized by marked increases in plasma LDL, including the LDL subfraction lipoprotein-X (Lp-X). To investigate the mechanisms underlying this paradox, we first examined the propensity to oxidation of unfractionated LDL isolated from PBC patients. After prolonged incubation with copper, PBC-LDL failed to increase the oxidation index or electrophoretic mobility noted in control LDL. An admixture of PBC-LDL or Lp-X with control LDL prevented oxidation of the latter in a dose-dependent manner. PBC-LDL was also noncompetitive against copper-oxidized LDL (oxLDL) for binding with a murine monoclonal anti-oxLDL antibody in a competitive ELISA. OxLDL exerts its proapoptotic and antiangiogenic effects in part by inhibiting fibroblast growth factor 2 (FGF2) expression. Preincubation of oxLDL with PBC-LDL, but not control LDL, attenuated the inhibitory effects of oxLDL on FGF2 expression in cultured bovine aortic endothelial cells (ECs). The antioxidant and prosurvival properties of PBC-LDL diminished after the patients underwent orthotopic liver transplantation. These results suggest that Lp-X reduces LDL atherogenicity by preventing LDL oxidation to protect EC integrity in the presence of hypercholesterolemia. They also suggest that altering LDL composition may be as important as reducing LDL concentration in preventing or treating atherosclerosis.     

Human lectin-like oxidized low-density lipoprotein receptor-1 functions as a dimer in living cells

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a unique scavenger receptor that plays important roles in atherogenesis and has been thought to function as a monomer. Using coimmunoprecipitation studies, we demonstrate that human LOX-1 (hLOX-1) forms constitutive homo-interactions in vivo. Western blot analysis of cell lysates under nonreducing or reducing conditions revealed one clear immunoreactive species corresponding to the size of a putative receptor dimer or a monomer, respectively, consistent with the presence of disulfide-linked hLOX-1 complexes. Site-directed mutagenesis studies indicated that cysteine 140 has a key role in the formation of these disulfide-linked hLOX-1 dimers. Eliminating this intermolecular disulfide bond markedly impairs the recognition of Escherichia coli by hLOX-1. Furthermore, these dimers can act as a "structural unit" to form noncovalently associated oligomers, as demonstrated by a membrane-impermeant crosslinker, which resulted in immunoreactive species corresponding to the sizes of putative tetramers and hexamers. These results provide the first evidence for the existence of hLOX-1 dimers/oligomers.     

Adsorption of low-density lipoprotein,its oxidation,and subsequent binding of specific recombinant antibodies: An in situ ellipsometric study

Background

Low-density lipoprotein (LDL) particles accumulate in the arterial wall and become oxidized during atherogenesis, leading to the formation of atherosclerotic plaques. The major protein of the LDL particle, apolipoprotein B-100 (apoB-100), becomes fragmented during oxidation and a target for the immune system.

Methods

In this study we used in situ ellipsometry to monitor the adsorption of LDL to solid silica surfaces and the effects of oxidation on the structure of the adsorbed LDL layer. We additionally investigated the binding kinetics of two recombinant human antibodies with different specificities recognizing epitopes of apoB-100 in surface-bound native and CuCl₂-oxidized LDL (oxLDL). The latter process was studied by adsorbing LDL and then adding the antibody and CuCl₂ while continuously monitoring adsorbed amount and the thickness of the film. The molar ratios between the antibodies and surface-bound LDL and oxLDL were calculated from these data.

Results

Our results indicate that oxidation of surface-bound LDL induces swelling of the layer, accompanied by a slight desorption. We further found that both antibodies were able to recognize LDL and oxLDL in its adsorbed orientation. Quantitative information was obtained on the number of available binding sites on surface-bound LDL and oxLDL for these two antibodies.

General significance

Using ellipsometry for real-time monitoring of adsorption, in situ oxidation of LDL and binding of specific recombinant antibodies to surface-bound LDL, will open up possibilities to map different conformations and orientations of LDL in the adsorbed state.