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.     

Effects of flow on LOX-1 and oxidized low-density lipoprotein interactions in brain endothelial cell cultures

Fluid shear stress and uptake of oxidized low-density lipoprotein (ox-LDL) into the vessel wall both contribute to atherosclerosis, but the relationship between shear stress and ox-LDL uptake is unclear. We examined the effects of flow, induced by orbital rotation of bEnd.3 brain endothelial cell cultures for 1 wk, on ox-LDL receptor (LOX-1) protein expression, ox-LDL uptake and ox-LDL toxicity. Orbitally rotated cultures showed no changes in LOX-1 protein expression, ox-LDL uptake or ox-LDL toxicity, compared to stationary cultures. Flow alone does not modify ox-LDL/LOX-1 signaling in bEnd.3 brain endothelial cells in vitro, suggesting that susceptibility of atheroprone vascular sites to lipid accumulation is not due solely to effects of altered flow on endothelium.     

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.     

LOX-1 pathway affects the extent of myocardial ischemia-reperfusion injury

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was originally identified as a receptor for oxidized low-density lipoprotein predominantly expressed in endothelial cells. LOX-1 expression can be induced in cardiomyocytes and that activation of LOX-1 is involved in apoptosis. To investigate possible roles of LOX-1 in myocardial ischemia-reperfusion injury, rats were subjected to coronary artery ligation for 1h followed by reperfusion for 2h. Immunohistochemistry revealed that expression of LOX-1 in cardiac myocytes was induced following ischemia-reperfusion but not ischemia alone. Administration of anti-LOX-1 monoclonal antibody resulted in a nearly 50% reduction in myocardial infarction size compared with that of normal IgG or saline (P<0.05). These findings suggest that activation of the LOX-1 pathway is involved in determining the extent of myocardial ischemia-reperfusion injury and that inhibition of the LOX-1 pathway may provide a novel strategy for treatment of acute myocardial infarction in humans.     

Molecular mechanism of statin-mediated LOX-1 inhibition

Statins are largely used in clinics in the treatment of patients with cardiovascular diseases for their effect on lowering circulating cholesterol. Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for ox-LDL, plays a central role in the pathogenesis of atherosclerosis and cardiovascular disorders. We have recently shown that chronic exposure of cells to lovastatin disrupts LOX-1 receptor cluster distribution in plasma membranes, leading to a marked loss of LOX-1 function. Here we investigated the molecular mechanism of statin-mediated LOX-1 inhibition and we demonstrate that all tested statins are able to displace the binding of fluorescent ox-LDL to LOX-1 by a direct interaction with LOX-1 receptors in a cell-based binding assay. Molecular docking simulations confirm the interaction and indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1. Classical molecular dynamics simulation technique applied to the LOX-1 CTLD, considered in the entire receptor structure with or without a statin ligand inside the tunnel, indicates that the presence of a ligand largely increases the dimer stability. Electrophoretic separation and western blot confirm that different statins binding stabilize the dimer assembly of LOX-1 receptors in vivo. The simulative and experimental results allow us to propose a CTLD clamp motion, which enables the receptor-substrate coupling. These findings reveal a novel and significant functional effect of statins.     

Enhanced radical scavenging activity of a procyanidin B3 analogue comprised of a dimer of planar catechin

Proanthocyanidins are oligomers of catechins that exhibit potent antioxidative activity and inhibit binding of oxidized low-density lipoprotein (OxLDL) to the lectin-like oxidized LDL receptor (LOX-1), which is involved in the onset and development of arteriosclerosis. Previous attempts aimed at developing proanthocyanidin derivatives with more potent antioxidative activity and stronger inhibition for LOX-1 demonstrated the synthesis of a novel proanthocyanidin derivative (1), in which the geometry of one catechin molecule in procyanidin B3 was constrained to a planar orientation. The radical scavenging activity of 1 was 1.9-fold higher than that of procyanidin B3. Herein, we synthesized another procyanidin B3 analogue (2), in which the geometries of both catechin molecules in the dimer were constrained to planar orientations. The radical scavenging activity of 2 was 1.5-fold higher than that of 1, suggesting that 2 may be a more effective candidate than 1 as a therapeutic agent to reduce oxidative stress induced in arteriosclerosis or related cerebrovascular disease.     

Microglial LOX-1 reacts with extracellular HSP60 to bridge neuroinflammation and neurotoxicity

Chronic neurodegeneration is in part caused by a vicious cycle of persistent microglial activation and progressive neuronal cell loss. However, the driving force behind this cycle remains poorly understood. In this study, we used medium conditioned by necrotic differentiated-PC12 cells to confirm that damaged neurons can release soluble injury signals, including heat shock protein 60 (HSP60), to efficiently promote the neurotoxic cycle involving microglia. Since lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has previously been identified as a novel receptor for HSP60, we hypothesize that LOX-1 through binding to extracellular HSP60 promotes microglia-mediated neuroinflammation. In this study, we observed that LOX-1 expression is induced upon toxic microglial activation, and discovered that LOX-1 is necessary in microglia for sensing soluble neuronal injury signal(s) in the conditioned medium to induce generation of pro-inflammatory mediators (IL-1β, TNF-α, NO and ROS) that promote neurotoxicity. Employing a unique eukaryotic HSP60-overexpression method, we further demonstrated that extracellular HSP60 acts on microglial LOX-1 to boost the production of pro-inflammatory factors (IL-1β, NO and ROS) in microglia and to propagate neuronal damage. These results indicate that LOX-1 is essential in microglia for promoting an inflammatory response in the presence of soluble neuronal-injury signals such as extracellular HSP60, thereby linking neuroinflammation and neurotoxicity.     

Oxidized LDL through LOX-1 modulates LDL-receptor expression in human coronary artery endothelial cells

Experimental studies have shown that oxidized low-density lipoprotein (ox-LDL) up-regulates its receptor LOX-1. Both ox-LDL and LOX-1 are expressed in atherosclerotic plaques. Native LDL concentrations are elevated in atherosclerosis, suggesting a reduction in LDL-receptors. We hypothesized that ox-LDL via LOX-1 could influence the expression of LDL-receptors. This study was designed to examine the interaction between ox-LDL, LOX-1, and LDL-receptors in human coronary artery endothelial cells (HCAECs). HCAECs were incubated with ox-LDL (10-80 microg/ml) for 3-24h. Ox-LDL decreased the expression of LDL-receptor in a concentration- and time-dependent fashion. The effects of ox-LDL were mediated by its endothelial receptor LOX-1, since pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92, 10 microg/ml) prevented the effect of ox-LDL on LDL-receptor expression. The role of LOX-1 was further confirmed by the use of an antisense to LOX-1 mRNA, which also blocked the effect of ox-LDL in LDL-receptor expression. In other experiments, ox-LDL as expected induced superoxide anion generation; and pretreatment of HCAECs with the anti-oxidants trolox and alpha-tocopherol (each 10 microM) inhibited the formation of superoxide anions as well as the down-regulation of LDL-receptor in response to ox-LDL. These studies provide the first evidence that ox-LDL via LOX-1 modulates LDL-receptor expression in HCAECs. The generation of free radicals elicited by ox-LDL may be a key step in this process.     

MicroRNA-328 ameliorates oxidized low-density lipoprotein-induced endothelial cells injury through targeting HMGB1 in atherosclerosis

Atherosclerosis has been recognized as a chronic inflammatory disease, which can harden the vessel wall and narrow the arteries. MicroRNAs exhibit crucial roles in various diseases including atherosclerosis. However, so far, the role of miR-328 in atherosclerosis remains barely explored. Therefore, our study concentrated on the potential role of miR-328 in vascular endothelial cell injury during atherosclerosis. In our current study, we observed that oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) apoptosis and inhibited cell viability dose-dependently and time-dependently. In addition, indicated dosage of ox-LDL obviously triggered HUVECs inflammation and oxidative stress process. Then, it was found that miR-328 in HUVECs was reduced by ox-LDL. HUVECs apoptosis was greatly repressed and cell survival was significantly upregulated by overexpression of miR-328. Furthermore, mimics of miR-328 rescued cell inflammation and oxidative stress process induced by ox-LDL. Oppositely, inhibitors of miR-328 strongly promoted ox-LDL-induced endothelial cells injury in HUVECs. By using bioinformatics analysis, high-mobility group box-1 (HMGB1) was predicted as a downstream target of miR-328. HMGB1 has been reported to be involved in atherosclerosis development. The correlation between miR-328 and HMGB1 was validated in our current study. Taken these together, it was implied that miR-328 ameliorated ox-LDL-induced endothelial cells injury through targeting HMGB1 in atherosclerosis.     

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.     

The hydrophobic tunnel present in LOX-1 is essential for oxidized LDL recognition and binding

Lectin-like oxidized LDL (ox-LDL) receptor-1 (LOX-1) is a type-II transmembrane protein that belongs to the C-type lectin family of molecules. LOX-1 acts as a cell surface endocytosis receptor and mediates the recognition and internalization of ox-LDL by vascular endothelial cells. Internalization of ox-LDL by LOX-1 results in a number of pro-atherogenic cellular responses implicated in the development and progression of atherosclerosis. In an effort to elucidate the functional domains responsible for the binding of ox-LDL to the receptor, a series of site-directed mutants were designed using computer modeling and X-ray crystallography to study the functional role of the hydrophobic tunnel present in the LOX-1 receptor. The isoleucine residue (I(149)) sitting at the gate of the channel was replaced by phenylalanine, tyrosine, or glutamic acid to occlude the channel opening and restrict the docking of ligands to test its functional role in the binding of ox-LDL. The synthesis, intracellular processing, and cellular distribution of all mutants were identical to those of wild type, whereas there was a marked decrease in the ability of the mutants to bind ox-LDL. These studies suggest that the central hydrophobic tunnel that extends through the entire LOX-1 molecule is a key functional domain of the receptor and is critical for the recognition of modified LDL.     

Specific Kv1.3 blockade modulates key cholesterol-metabolism-associated molecules in human macrophages exposed to ox-LDL

Cholesterol-metabolism-associated molecules, including scavenger receptor class A (SR-A), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), CD36, ACAT1, ABCA1, ABCG1, and scavenger receptor class B type I, can modulate cholesterol metabolism in the transformation from macrophages to foam cells. Voltage-gated potassium channel Kv1.3 has increasingly been demonstrated to play an important role in the modulation of macrophage function. Here, we investigate the role of Kv1.3 in modulating cholesterol-metabolism-associated molecules in human acute monocytic leukemia cell-derived macrophages (THP-1 macrophages) and human monocyte-derived macrophages exposed to oxidized LDL (ox-LDL). Human Kv1.3 and Kv1.5 channels (hKv1.3 and hKv1.5) are expressed in macrophages and form a heteromultimeric channel. The hKv1.3-E314 antibody that we had generated as a specific hKv1.3 blocker inhibited outward delayed rectifier potassium currents, whereas the hKv1.5-E313 antibody that we had generated as a specific hKv1.5 blocker failed. Accordingly, the hKv1.3-E314 antibody reduced percentage of cholesterol ester and enhanced apoA-I-mediated cholesterol efflux in THP-1 macrophages and human monocyte-derived macrophages exposed to ox-LDL. The hKv1.3-E314 antibody downregulated SR-A, LOX-1, and ACAT1 expression and upregulated ABCA1 expression in THP-1 macrophages and human monocyte-derived macrophages. Our results reveal that specific Kv1.3 blockade represents a novel strategy modulating cholesterol metabolism in macrophages, which benefits the treatment of atherosclerotic lesions.     

LOX-1在D-葡萄糖诱导人肾小球系膜细胞表达TGF-β1中的作用

目的探讨血凝素样氧化低密度脂蛋白受体1（LOX-1）在D-葡萄糖诱导人肾小球系膜细胞表达转化生长因子β1（TGF-β1）中的作用。方法在体外培养人肾小球系膜细胞,在不同时间加入不同浓度的D-葡萄及LOX-1特异性阻滞剂JTX92,用半定量RT-PCR法检测LOX-1和TGF-β1基因表达的相对含量,用Western blot法检测p38 MAPK蛋白质的相对含量,用酶联免疫吸附法（ELISA）检测细胞培养液中TGF-β1浓度。结果D-葡萄糖以时间和浓度依赖的方式增加细胞内LOX-1和TGF-β1 mRNA表达和培养液中TGF-β1浓度,同时也以时间和浓度依赖的方式增加p38 MAPK的表达,JTX92可以明显抑制LOX-1、TGF-β1和p38 MAPK的表达。结论高浓度D-葡萄糖可能通过上调LOX-1的表达,激活细胞内的p38 MAPK信号传递途径,促使人肾小球系膜细胞合成并分泌大量TGF-β1,参与糖尿病肾病的发生发展。     

The lectin-like oxidized low-density-lipoprotein receptor: a pro-inflammatory factor in vascular disease

Scavenger receptors are membrane glycoproteins that bind diverse ligands including lipid particles, phospholipids, apoptotic cells and pathogens. LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is increasingly linked to atherosclerotic plaque formation. Transgenic mouse models for LOX-1 overexpression or gene knockout suggests that LOX-1 contributes to atherosclerotic plaque formation and progression. LOX-1 activation by oxidized LDL (low-density lipoprotein) binding stimulates intracellular signalling, gene expression and production of superoxide radicals. A key question is the role of leucocyte LOX-1 in pro-atherogenic lipid particle trafficking, accumulation and signalling leading to differentiation into foam cells, necrosis and plaque development. LOX-1 expression is elevated within vascular lesions and a serum soluble LOX-1 fragment appears diagnostic of patients with acute coronary syndromes. LOX-1 is increasingly viewed as a vascular disease biomarker and a potential therapeutic target in heart attack and stroke prevention.     

Advanced glycation end products serve as ligands for lectin-like oxidized low-density lipoprotein receptor-1(LOX-1): biochemical and binding characterizations assay

Advanced glycation end products (AGEs) are a class of complex heterogeneous compounds which accumulate with age and is known to be involved in the pathogenesis of several diseases from diabetes to atherosclerosis. AGEs serve as ligands for multiple receptors including scavenger receptor (SR-A), CD36, and SR-BIota. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays an important role in both atherosclerosis and is found to be an endothelial cell receptor for AGEs. To explore the binding characterization of AGEs to LOX-1, AGEs were prepared by three different reducing sugars (d-glucose, d-fructose, and d-ribose) and the biochemical characterization including, free amino groups, free amine content, fructosamine residues, carbonyl content, fluorescence, and absorbance were determined. The binding activity was determined by FITC labeled AGEs using Chinese hamster ovary-K1 cells stably transfected with human LOX-1 gene. The obtained AGEs showed significant differences in the extent of side chain modifications, carbonyl content, fluorescence, and absorption models. All of the AGEs showed specific and saturable binding to hLOX-1-CHO-K1 cells. Furthermore, dose-dependent binding processes were observed. However, the maximal cellular binding of AGEs differs between the sugars (glucose > ribose > fructose). In addition, oxidized low-density lipoprotein (ox-LDL) could significantly inhibit the binding of AGEs to LOX-1 with different inhibitory efficiency. LOX-1 serves as receptor for AGEs which may give some insight into the role of LOX-1 in the pathogenesis of diabetes and related disorders.     

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

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)-gamma was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-gamma 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-gamma and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.