Binding Of Oxidized LDL Research Articles

“Affimer” synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p< 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

Protamine may have anti-atherogenic potential by inhibiting the binding of oxidized-low density lipoprotein to LOX-1.

Oxidized low-density lipoprotein (ox-LDL) leads to atherosclerosis via lectin-like oxidized lipoprotein receptor-1 (LOX-1), one of the major receptor for ox-LDL. Inhibition of the binding of ox-LDL to LOX-1 decreases the proinflammatory and atherosclerotic events. The aim of the present study was to investigate whether protamine, a polybasic nuclear protein, interferes the binding of ox-LDL to LOX-1. Using sandwich ELISA with newly generated antibody, we measured the blocking effect of protamine on the binding of ox-LDL to LOX-1. Protamine dose-dependently inhibited the binding of ox-LDL to LOX-1. DiI-labeled ox-LDL uptake assay in two types of cultured human endothelial cells was performed with fluorescence microplate reader. Activation of extracellular-signal-regulated kinase (ERK)1/2 by ox-LDL was analyzed by immunoblotting. We found that protamine suppressed uptake of ox-LDL in endothelial cells and inhibited ERK1/2 activation by ox-LDL. These results suggest that protamine may possess anti-atherogenic potential by inhibiting ox-LDL binding to LOX-1 through electrostatic interactions.

DOPG small unilamellar vesicles function as nano-carriers targeting the clustered lectin-like oxidized LDL receptor (LOX-1) on the cell surface

The lectin-like oxidized low-density lipoprotein (OxLDL) receptor (LOX-1) is the primary OxLDL receptor of vascular endothelial cells. OxLDL binding to LOX-1 promotes atherogenesis by upregulating LOX-1 expression to further sensitize cells against OxLDL. In this work, we demonstrate that small unilamellar vesicles (SUVs) composed of the phospholipid DOPG (1,2-dioleyl-sn-glycero-3-[phospho-rac-(1-glycerol)) (DOPG-SUV) inhibit the binding of acetylated LDL (AcLDL, a chemically stable surrogate for OxLDL) to LOX-1 on cells. DOPG-SUVs internalize into the cells expressing high levels of LOX-1 even in the presence of an excess amount of AcLDL. DOPG-SUV thus functions as a nano-carrier to deliver therapeutic agents selectively to cells in atherosclerotic lesions. The difference in the binding affinities between DOPG-SUV and AcLDL to LOX-1 clusters were quantitatively elucidated by surface plasmon resonance experiments using a sensor harboring the LOX-1 extracellular domains doped on self-assembled monolayers, which mimics the presence of LOX-1 clusters on cells. DOPG-SUV has a negatively charged surface and is larger in diameter than that of AcLDL. DOPG-SUVs interact with more receptors in the LOX-1 cluster than that envisaged for AcLDL or OxLDL binding, which explains the higher affinity of DOPG-SUV to the LOX-1 cluster when compared with those of modified LDLs.

Angiotensin II induces the aggregation of native and oxidized low-density lipoprotein.

Modifications of low-density lipoprotein (LDL), such as oxidation and aggregation, and angiotensin (Ang) peptides are involved in the pathogenesis of atherosclerosis. Here, we investigated the relationship between one of the Ang peptides, AngII, and two LDL modifications, oxidation and aggregation. Using polyacrylamide gel electrophoresis and aggregation assays, we noted that AngII markedly induced the aggregation of LDL and oxidized LDL (Ox-LDL), and bound to both the aggregated and non-aggregated forms. In contrast, a peptide (AngIII) formed by deletion of N-terminal Asp of AngII induced the aggregation of Ox-LDL but not LDL. From tyrosine fluorescence measurements, we noted that AngII interacted with two major lipid components in LDL and Ox-LDL, phosphatidylcholine (PC) and oxidized PC, while AngIII interacted with oxidized PC, but not with PC and lysophosphatidylcholine. Moreover, results from thiobarbituric acid-reactive substances assay proved that AngII did not induce oxidation of LDL. These results suggest that AngII can be involved in the pathogenesis of atherosclerosis by binding to LDL and Ox-LDL-especially to the major lipid components, PC and oxidized PC-followed by inducing the aggregation of LDL and Ox-LDL and that the N-terminal Asp of AngII is important for the binding and aggregation specificity of LDL and Ox-LDL.

Development and application of a nonradioactive binding assay of oxidized low-density lipoprotein to macrophage scavenger receptors

Macrophages play a key role in atherogenesis in part through excessive uptake of oxidized LDL (OxLDL) via scavenger receptors. Binding of OxLDL to macrophages has traditionally been assessed using radiolabeled OxLDL. To allow more efficient and convenient measurements, we developed a nonradioactive binding assay in which biotinylated OxLDL (Bt-OxLDL) is added to macrophages in 96-well microtiter culture plates under various conditions and the extent of binding is determined using solid phase chemiluminescent immunoassay techniques. As examples, we show that Bt-OxLDL displayed high and saturable binding to macrophages in contrast to Bt-LDL, which showed very low binding. In competition assays, unlabeled OxLDL and the anti-OxLDL monoclonal antibody E06 inhibited Bt-OxLDL binding to macrophages in a dose-dependent manner. Specific binding of Bt-OxLDL to ApoE/SR-A/CD36 triple knockout macrophages was reduced by 80% as compared with binding to macrophages from ApoE knockout mice. Binding of Bt-OxLDL to CD36 transfected COS-7 cells showed enhanced saturable binding compared with mock-transfected cells. This assay avoids the use of radioactivity and uses small amounts of materials. It can be used to study binding of OxLDL to macrophages and factors that influence this binding. The techniques described should be readily adaptable to study of other ligands, receptors, and cell types.

Native High Density Lipoproteins (HDL) Interfere with Platelet Activation Induced by Oxidized Low Density Lipoproteins (OxLDL)

Platelets and lipoproteins play a crucial role in atherogenesis, in part by their ability to modulate inflammation and oxidative stress. While oxidized low density lipoproteins (OxLDL) play a central role in the development of this disease, high density lipoproteins (HDL) represent an atheroprotective factor of utmost importance. As platelet function is remarkably sensitive to the influence of plasma lipoproteins, it was the aim of this study to clarify if HDL are able to counteract the stimulating effects of OxLDL with special emphasis on aspects of platelet function that are relevant to inflammation. Therefore, HDL were tested for their ability to interfere with pro-thrombotic and pro-inflammatory aspects of platelet function. We are able to show that HDL significantly impaired OxLDL-induced platelet aggregation and adhesion. In gel-filtered platelets, HDL decreased both the formation of reactive oxygen species and CD40L expression. Furthermore, HDL strongly interfered with OxLDL-induced formation of platelet-neutrophil aggregates in whole blood, suggesting that platelets represent a relevant and sensitive target for HDL. The finding that HDL effectively competed with the binding of OxLDL to the platelet surface might contribute to their atheroprotective and antithrombotic properties.

Radioiodinated peptide probe for selective detection of oxidized low density lipoprotein in atherosclerotic plaques

Despite the significant effort in developing radioprobes for atherosclerosis, few have low molecular weight. Oxidized LDL (OxLDL), a highly proinflammatory and proatherogenic factor that is abundant in atherosclerotic plaques, plays a pivotal role in plaque destabilization, which makes OxLDL a relevant probe target. We developed a radioiodinated short peptide, AHP7, as a low molecular weight probe for specific OxLDL imaging and evaluated its utility using myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits (WHHLMI). [¹²⁵I]AHP7 was designed and synthesized based on the sequence of Asp-hemolysin, an OxLDL binding protein extracted from Aspergillus fumigatus. In vitro binding studies with OxLDL having varying degrees of oxidation were performed. Radioactivity accumulation in the aorta was measured 30 min post-administration in rabbits. Autoradiography and histological studies were performed using serial aorta sections. A radioiodinated scrambled peptide ([¹²⁵I]AHP scramble) was used as a negative control. [¹²⁵I]AHP7 bound to OxLDL in proportion to the degree of oxidation (R=0.91, P<0.0001) and was inhibited by unlabeled AHP7 in a concentration-dependent manner. The aorta accumulation level and aorta/blood and aorta/muscle ratios of [¹²⁵I]AHP7 in WHHLMI were 2.8-, 1.3- and 1.8-fold higher, respectively, than those in control rabbits (P<0.001). Co-administration of AHP7 significantly reduced [¹²⁵I]AHP7 radioactivity in aorta sections (P<0.0001). Regional radioactivity levels in the aorta sections showed nonuniformity but similarity to the immunohistochemical OxLDL density. The potential of radioiodinated AHP7 for selectively imaging OxLDL was demonstrated both in vitro and in vivo.

Abstract P097: Endothelial Overexpression of LOX-1 Decreases Arterial Thrombosis and TF Expression in Vivo : Role of SIRT-1 and NFκB

Background -The hallmark of the initiation of atherosclerotic lesion is foam cell formation, and oxidized LDL (OxLDL) is believed to play a key role in the initiation of the atherosclerotic process. OxLDL is internalized by several receptors, such as SR-AI/II, SR-BI, CD36, and CD68. OxLDL is also internalized by endothelial cells, but this uptake depends on receptors other than the classic scavenger receptors. In 1997, a lectin-like oxidized LDL receptor-1 (LOX-1, OLR1) was identified in bovine aortic endothelial cells. LOX- 1 is a type II membrane glycoprotein with an apparent molecular weight of 50 kDa. It has a C-terminal extracellular C-type lectin-like domain. This lectin-like domain is essential for binding to OxLDL. Binding of OxLDL to LOX-1 induces several cellular events in endothelial cells, such as activation of transcription factor NF-kB, upregulation of MCP-1, and reduction in intracellular NO, which may trigger the onset of cardiovascular events or accelerate the development of atherosclerosis. Methods and Results - We generated endothelial-specific LOX-1 transgenic mice using the Tie2 promoter ( LOX-1TG ). 12-week-old male LOX-1TG and wild-type (WT) mice were applied for carotid artery thrombosis model. LOX-1TG mice developed carotid artery thrombosis within a mean occlusion time of 36.96±4.83 min, while WT control mice occluded within a mean time period of 22.75±3.87 min (n=10, P &lt; 0.05). Initial blood flow in carotid artery did not differ between both groups of mice. Decreased occlusion time was in LOX-1TG mice vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression, macrophage accumulation and aortic fatty streaks were increased, while eNOS phosphorylation and endothelial function were reduced. In endothelial cells of LOX-1TG mice, reactive oxygen species were increased and the transcription factors NF-κB and Oct-1 activated. In atherosclerotic LOX-1TG/ApoE −/− mice,high cholesterol diet increased VCAM-1 expression, number of macrophages, T-cells as well as plaque size. Conclusions -Thus, our data suggest that LOX-1 plays a protective role in the arterial thrombosis when expressed at unphysiological levels. Therefore, LOX-1 might represent a novel therapeutic target for atherosclerosis.

Atherosclerosis development in SLE patients is not determined by monocytes ability to bind/endocytose Ox-LDL

Patients with systemic lupus erythematosus (SLE) have a high risk of developing cardiovascular disease; however, the mechanisms involved in the early onset of atherosclerosis in these patients are not clear. Scavenger receptors, CD36 and CD163 are expressed by mononuclear phagocytes and participate in the binding and uptake of oxidized low-density lipoproteins (Ox-LDL), contributing to foam-cells formation and atherosclerosis development. The aim of the present study was to evaluate CD36+ and CD163+ expression and Ox-LDL removal by monocytes from SLE and atherosclerotic patients, compared to similar age-range healthy controls. Healthy controls, SLE, and atherosclerotic patients were evaluated for carotid intima media thickness (CIMT), lipid profile, and native LDL (N-LDL) and Ox-LDL binding/endocytosis. SLE patients presented decreased high-density lipoproteins (HDL) and increased Triglyceride levels, and half of the SLE patients had increased CIMT, compared to their healthy controls (HCSLE). The number of CD14+CD163+ cells was increased in atherosclerosis healthy controls (HCAtheros) compared to HCSLE, but there were no differences between SLE or atherosclerotic patients and their respective healthy controls. Clearance assays revealed a similar capacity to bind/endocytose Ox-LDL by monocytes from SLE patients and HCSLE, and an increased binding and endocytosis of Ox-LDL by monocytes from atherosclerotic patients, compared to HCAtheros. The decreased CD36 and CD163 expression observed in atherosclerotic and SLE patients, respectively, suggest that these inflammatory conditions modulate these receptors differentially. The increased CIMT observed in SLE patients cannot be explained by Ox-LDL binding/endocytosis, which was comparable to their controls.

Oxidized Low-Density Lipoprotein and Atherosclerosis

Chance and serendipity play major roles in the history of science. Too often, though, their contributions do not show up in formal publications. We all tend to shape history according to the styles of the times and according to our own biases. Acknowledged or unacknowledged, there is a tendency to want the scientific “story” to be neat and more or less linear. The genesis of the oxidized low-density lipoprotein (OxLDL) hypothesis owed a great deal to happenstance, but that may not be readily apparent from the formal articles. We take this opportunity to tell the whole story, at least as we remember it. Several apparently unrelated events occurring at about the same time in Oslo, Norway; in Cleveland, Ohio; in La Jolla, California; in New York, New York; and in Dallas, Texas converged to lay the groundwork for the hypothesis that oxidative modification of LDL might be important in atherogenesis. Later developments regarding the relationship between OxLDL and the immune system in atherogenesis, again, were often smiled on by chance and serendipity. In 1979, a young Norwegian researcher, Tore Henriksen, got in touch with our laboratory asking whether he could come to La Jolla to learn more about lipoproteins. He and his colleagues in Oslo had observed that under certain conditions, LDL was highly toxic for endothelial cells in culture, leading to cell death in just 24 hours.1 Almost simultaneously and independently, Chisolm and coworkers in Cleveland made very similar observations.2 Both groups noted that addition of whole serum or purified high-density lipoprotein to the medium completely prevented the toxic effects of the LDL, but the mechanism(s) involved remained unclear. Henriksen arrived in La Jolla in the summer of 1980 and demonstrated to us how drastically LDL affected the cultured cells. We agreed that the phenomenon was worth …

Surface plasmon resonance study on functional significance of clustered organization of lectin-like oxidized LDL receptor (LOX-1)

Lectin-like oxidized low-density lipoprotein (OxLDL) receptor 1 (LOX-1) is the major OxLDL receptor of vascular endothelial cells and is involved in an early step of atherogenesis. LOX-1 exists as a disulfide-linked homodimer on the cell surface, which contains a pair of the ligand-binding domains (CTLD; C-type lectin-like domain). Recent research using living cells has suggested that the clustered state of LOX-1 dimer on the cell is functionally required. These results questioned how LOX-1 exists on the cell to achieve OxLDL binding. In this study, we revealed the functional significance of the clustered organization of the ligand-binding domain of LOX-1 with surface plasmon resonance. Biotinylated CTLD was immobilized on a streptavidin sensor chip to make CTLD clusters on the surface. In this state, the CTLD had high affinity for OxLDL with a dissociation constant ( K D) in the nanomolar range. This value is comparable to the K D measured for LOX-1 on the cell. In contrast, a single homodimeric LOX-1 extracellular domain had lower affinity for OxLDL in the supra-micromolar range of K D. Monomeric CTLD showed marginal binding to OxLDL. In combination with the analyses on the loss-of-binding mutant W150A, we concluded that the clustered organization of the properly formed homodimeric CTLD is essential for the strong binding of LOX-1 to OxLDL.

Induction of hypertrophic chondrocyte-like phenotypes by oxidized LDL in cultured bovine articular chondrocytes through increase in oxidative stress

It has been reported that the lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor 1 (LOX-1) is expressed by chondrocytes in osteoarthritis (OA) cartilage and that Ox-LDL binding to LOX-1 increases intracellular oxidative stress in cultured bovine articular chondrocytes (BACs). It was recently demonstrated that reactive oxygen species (ROS) induce hypertrophic differentiation of chondrocytes in the growth plate. It has also been shown that activated chondrocytes in OA have hypertrophic chondrocyte-like phenotypes. The purpose of this study was to determine whether Ox-LDL induces hypertrophic chondrocyte-like phenotypes in BACs. Changes in type X collagen (COL10) and runt-related transcription factor 2 (Runx2) mRNA expression in BACs after Ox-LDL stimulation were investigated using real-time polymerase chain reaction (PCR). Western blotting and immunofluorescent cell staining were used to investigate changes in protein level. The antioxidant N-acetyl cysteine (NAC) was used to ascertain whether oxidative stress is involved in COL10 and Runx2 expression. We induced LOX-1 knockdown cells using small interfering RNA (siRNA) to examine the receptor specificity of Ox-LDL. COL10 expression was upregulated by Ox-LDL in a time- and dose-dependent manner. Immunofluorescent staining showed that Ox-LDL increased COL10 production in the extracellular matrix. Ox-LDL-induced upregulation of COL10 was suppressed by pretreatment with NAC and siRNA. Expression of Runx2 was upregulated by Ox-LDL and H(2)O(2), and these effects were suppressed by NAC pretreatment. Ox-LDL binding to LOX-1 induces a hypertrophic chondrocyte-like phenotype through oxidative stress, indicating that Ox-LDL plays a role in the degeneration of cartilage.

Oligomerization is required for the activity of recombinant soluble LOX‐1

LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.

Oxidized LDL binding to LOX-1 enhances MCP-1 expression in cultured human articular chondrocytes

It has been suggested that oxidized low-density lipoprotein (ox-LDL) has some roles in progression of osteoarthritis. The purpose of this study is to investigate whether ox-LDL binding to lectin-like ox-LDL receptor 1 (LOX-1) enhances monocyte chemoattractant protein 1 (MCP-1) expression in cultured human articular chondrocytes (HACs). The time course and dose response of MCP-1 mRNA expression and MCP-1 protein release into medium following ox-LDL stimulation were investigated using quantitative Real time PCR (delta-delta Ct method) and enzyme-linked immunosorbent assay (ELISA), respectively. To examine the receptor specificity of ox-LDL action, HACs were preincubated with anti-human LOX-1 monoclonal antibody (TS92). A time-course study revealed that MCP-1 mRNA expression increased 5.09+/-0.86 fold 12h after ox-LDL stimulation compared to time-0. ox-LDL stimulation increased MCP-1 protein level in conditioned medium in a time-dependent manner. Increased MCP-1 level was evident 6h after stimulation, reaching 830+/-91 pg/ml at 24h (33+/-8 pg/ml at time-0). Dose responses of MCP-1 expression were also evident in mRNA and protein levels. Pretreatment with TS92 markedly suppressed these stimulating effects of ox-LDL, although that with non-specific IgG did not. Native LDL did not affect MCP-1 expression. Our results suggest that ox-LDL enhances MCP-1 expression in HACs and supports the hypothesis that ox-LDL is involved in cartilage degeneration.

CD36 N-terminal cytoplasmic domain is not required for the internalization of oxidized low-density lipoprotein

The uptake of OxLDLs (oxidized low density lipoproteins) by CD36-expressing macrophages in the arterial intima and the subsequent 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. The present study has addressed the function of the CD36 N-terminal cytoplasmic domain in the binding and internalization of OxLDL. A selection of CD36 N-terminal cytoplasmic domain mutants were generated and stably expressed in HEK-293 (human embryonic kidney) cells. The capacity of three mutants [CD36_C3/7-A (CD36-C3A/C7A), CD36_D4/R5-A (CD36-D4A/R5A) and CD36_nCPD(-) (CD36 lacking the N-terminal cytoplasmic domain)] to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluorimetry. Each of the CD36 constructs was expressed at differing levels at the cell surface, as measured by flow cytometry and Western blotting. Following incubation with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-OxLDL, cells bearing the CD36_wt (wild-type CD36), CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD(-) constructs all internalized DiI-OxLDL into endosomal structures, whereas empty-vector-transfected cells failed to do so, indicating that, unlike the C-terminal cytoplasmic domain, the N-terminal cytoplasmic domain is not essential for the endocytosis of OxLDL. In conclusion, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 N-terminal cytoplasmic domain. However, the N-terminal cytoplasmic domain may conceivably be implicated in the maturation of CD36.

Dietary homocysteine promotes atherosclerosis in apoE-deficient mice by inducing scavenger receptors expression

Elevated plasma homocysteine (Hcy) levels have been recognized as an independent risk factor for atherosclerosis leading to cardiovascular diseases. However, the mechanisms contributing to atherosclerosis have not been delineated. Since, scavenger receptors mediated uptake of oxidized-LDL (oxLDL) by macrophages resulting in foam cell formation is an early event in atherosclerosis, we hypothesized that atherogenic effects of Hcy may be mediated via regulating expression of scavenger receptor(s). We have tested this hypothesis using apoE−/− female mice fed normal rodent chow (NC) diet or NC supplemented with Hcy in drinking water (9g/L). Hcy-fed mice showed increased fatty streak lesions in aortic sinus/root compared to NC group without alterations in plasma lipid profiles. Similar findings were observed in the enface analysis of the descending aorta. To determine the molecular mechanisms underlying Hcy-mediated progression of fatty streak lesions, expression of scavenger receptors such as CD36 and lectin-like oxidized LDL binding protein-1 (LOX-1) in the aortic lesions were analyzed. Interestingly, Hcy-fed mice had increased immuno-positive staining for CD36 and LOX-1 in the atherosclerotic lesions compared to NC-fed mice. In vitro analyses showed neither Hcy nor HcyLDL directly affect the expression of CD36 and LOX-1 on mouse macrophages. However, Hcy supplementation in apoE−/− mice resulted in elevated oxLDL levels in plasma. Since oxLDL has been shown to upregulate the expression of CD36 and LOX-1, these findings suggest that Hcy may exert its atherogenic effect in part by elevating the levels of oxLDL. Interestingly, interaction of monocytes with Hcy-activated endothelial cells resulted in upregulation of CD36 expression on monocytes, suggesting a possible mechanism by which Hcy may upregulate CD36 expression at the lesion site. Further, these findings suggest a novel mechanism by which Hcy may promote atherogenesis.

Abstract 1454: Targeted Molecular Imaging Probes For In Vivo MR Detection Of Atherosclerotic Lesions Using Antibodies Against Oxidized Low Density Lipoprotein

Background: Oxidized low-density lipoprotein (OxLDL) plays a key role in the initiation, progression and destabilization of atherosclerotic plaque. Molecular imaging probes that target OxLDL may allow in vivo detection of vulnerable plaques. In this study, magnetic resonance imaging (MRI) was used to detect atherosclerotic lesions in apolipoprotein deficient mice (ApoE−/−) using micelles comprised of gadolinium lipids, fluorescent rhodamine, PEG-lipids, and MDA2, a murine monoclonal antibody that binds malondialdehyde (MDA) lysine epitopes present in OxLDL. Materials and Results: Untargeted micelles, MDA2-labeled micelles and nonspecific polyclonal IgG micelles were prepared and characterized with respect to OxLDL binding capacity, pharmacokinetics, and biodistribution in wild type (WT) and ApoE−/− mice. MR imaging was performed at 9.4T over a 3-week interval after administration of 0.075 mmol Gd/kg micelles. MDA2 increased the micelle size, blood half-life, and MR efficacy relative to untargeted and IgG-micelles. Maximal plaque enhancement (&gt;125%) was observed 72 hours post MDA2-micelle injection (Figure ). Untargeted and IgG-micelles did not exhibit significant wall enhancement at any of the time points studied. Confocal microscopy revealed that MDA2-micelles accumulate within foam cells associated with atherosclerotic plaque. WT mice showed no significant MR wall enhancement for any of the micelles studied. Conclusions: MR imaging using MDA2-micelles demonstrates specific targeting of OxLDL and foam cells and provides excellent MR image quality. This study suggests that it may be feasible to image similar atherosclerotic lesions in humans with MRI.

Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-γ

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-γ was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-γ inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-γ and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.

Binding Characterizations of Asp‐Hemolysin to Oxidized Low‐Density Lipoprotein

AbstractFor Abstract see ChemInform Abstract in Full Text.

Binding Characterizations of Asp-hemolysin to Oxidized Low-Density Lipoprotein

Oxidized low-density lipoprotein (Ox-LDL) is known to be involved in the generation and progression of atherosclerosis. Ox-LDL has a number of potentially atherogenic effects on vascular cells, including uncontrol uptake by scavenger receptors. Asp-hemolysin, a hemolytic toxin from Aspergillus fumigatus, is a binding protein for Ox-LDL. This study was undertaken to clarify the binding specificity of Asp-hemolysin to Ox-LDL. We examined the binding specificity of Asp-hemolysin to Ox-LDL using several modified lipoproteins and scavenger-receptor ligands. Asp-hemolysin bound to Ox-LDL with shorter LDL oxidation times. However, Asp-hemolysin did not bind to acetylated LDL. The native high-density lipoprotein (n-HDL) and modified HDL (e.g., acetylated HDL, oxidized HDL) also had no Asp-hemolysin binding. Inhibitors of scavenger-receptor binding, including maleylated bovine serum albumin, polyinosinic acid, dextran sulfate, and fucoidin, had no effect on the binding of Ox-LDL to Asp-hemolysin. Surface plasmon-resonance studies revealed that Ox-LDL binds with high affinity (K(D)=0.63 microg/ml) to Asp-hemolysin. Furthermore, we have shown that Ox-LDL strongly inhibits the hemolytic activity of Asp-hemolysin and that the removal of lysophosphatidylcholine (lysoPC) from Ox-LDL abolished the inhibition. We also investigated the interaction between Asp-hemolysin and lysoPC as a typical lipid moiety of Ox-LDL. The binding of Asp-hemolysin to LDL oxidized for different times depended on the lysoPC content in each Ox-LDL. In addition, the inhibition of lysoPC production in Ox-LDL by phenylmethylsulfonyl fluoride (PMSF) pretreatment of LDL resulted in a marked decrease in Asp-hemolysin binding to PMSF-pretreated Ox-LDL. The binding analysis of Asp-hemolysin to lysoPC revealed that Asp-hemolysin binds directly to lysoPC. We conclude that Asp-hemolysin is a specific binding protein with high affinity for Ox-LDL and that its binding specificity is distinct from any receptor for Ox-LDL.