Requirements of basic amino acid residues within the lectin-like domain of LOX-1 for the binding of oxidized low-density lipoprotein

AbstractLectin-like OxLDL receptor-1 (LOX-1) was identified as the major receptor for oxidized low-density lipoprotein (OxLDL) in aortic endothelial cells. LOX-1 is a type II membrane protein that structurally belongs to the C-type lectin family. Here, we found that the lectin-like domain of LOX-1 is essential for ligand binding, but the neck domain is not. In particular, the large loop between the third and fourth cysteine of the lectin-like domain plays a critical role for OxLDL binding as well as C-terminal end residues. Alanine-directed mutagenesis of the basic amino acid residues around this region revealed that all of the basic residues are involved in OxLDL binding. Simultaneous mutations of these basic residues almost abolished the OxLDL-binding activity of LOX-1. Electrostatic interaction between basic residues in the lectin-like domain of LOX-1 and negatively charged OxLDL is critical for the binding activity of LOX-1

Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) serves as an endothelial receptor for advanced glycation end products (AGE)

AbstractAdvanced glycation end products (AGE) are known to serve as ligands for the scavenger receptors such as SR-A, CD36 and SR-BI. In the current study, we examined whether AGE is recognized by lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). Cellular binding experiments revealed that AGE-bovine serum albumin (AGE-BSA) showed the specific binding to CHO cells overexpressing bovine LOX-1 (BLOX-1), which was effectively suppressed by an anti-BLOX-1 antibody. Cultured bovine aortic endothelial cells also showed the specific binding for AGE-BSA, which was suppressed by 67% by the anti-BLOX-1 antibody. Thus, LOX-1 is identified as a novel endothelial receptor for AGE

Nebivolol and its 4-keto derivative increase nitric oxide in endothelial cells by reducing its oxidative inactivation

AbstractObjectivesThe objective of the present study was to elucidate the vasodilator mechanisms of nebivolol, a high selective β1-receptor antagonist with antioxidant properties.BackgroundOxidative inactivation of nitric oxide (NO) is regarded as an important cause of its decreased biological activity.MethodsOxidative stress was induced through the binding of oxidized (ox)-low-density lipoprotein (LDL) to its specific endothelial receptor, called “lectin-like oxidized LDL receptor-1” (LOX-1), in bovine and human endothelial cells and in Chinese hamster ovary cells stably expressing bovine LOX-1 (BLOX-1-CHO cells). Reactive oxygen species (ROS), superoxide (O2·−), and NO were measured in cells by flow cytometry.ResultsNebivolol and its 4-keto derivative prevented in a dose-dependent manner the increase of ROS (p < 0.001) and O2·−(p < 0.001) in bovine aortic endothelial cells (BAECs), human umbilical vein endothelial cells (HUVECs), and BLOX-1-CHO cells stimulated with ox-LDL. Atenolol had no effect. The incubation of HUVECs and BAECs with ox-LDL reduced basal and bradykinin-induced NO and nitrite concentration (p from <0.001 to <0.01). Nebivolol and its 4-keto derivative prevented the reduction of basal and stimulated NO and nitrite concentration (p from <0.001 to <0.01) while atenolol had no effect. The preincubation of BAECs with blocking anti-LOX-1 monoclonal antibody (LOX-1 mAb) significantly counteracted the effect of ox-LDL on stimulated generation of NO (p < 0.001), but the effect was significantly lower than that of nebivolol and its 4-keto derivative alone (p < 0.01).ConclusionsIn conclusion, the findings of the present study indicate that nebivolol increases NO also by decreasing its oxidative inactivation

Endothelin-1 receptor blockade prevents renal injury in experimental hypercholesterolemia

Endothelin-1 receptor blockade prevents renal injury in experimental hypercholesterolemia.BackgroundThe potent vasoconstrictor endothelin-1 is involved in regulation of renal function, and is up-regulated in hypercholesterolemia (HC), a risk factor for renal disease that increases oxidative stress and impairs renal hemodynamic responses. However, the involvement of endothelin (ET) in this disease process is yet unknown.MethodsRegional renal hemodynamics and function in vivo were quantified in pigs at baseline and during infusion of acetylcholine using electron beam computed tomography after a 12-week normal diet (N = 6), HC diet (N = 6), and HC diet orally supplemented (4mg/kg/day) with the selective ET receptor-A (ET-A) blocker ABT-627 (HC+ET-A, N = 6). Plasma levels of 8-epi-PGF2-α-isoprostanes, markers of oxidative stress, were measured using enzyme immunoassay, and renal tissue was studied ex vivo using Western blotting, electrophoretic mobility shift assay, and immunohistochemistry.ResultsTotal and low-density lipoprotein (LDL) cholesterol were similarly increased, but isoprostanes were decreased in HC+ET-A compared to HC alone. Basal renal perfusion was similar among the groups, while glomerular filtration rate (GFR) increased in HC+ET-A compared to HC. Stimulated perfusion and GFR were blunted in HC, but normalized in HC+ET-A. Moreover, ET blockade increased expression of endothelial nitric oxide synthase, and decreased endothelial expression of the oxidized-LDL receptor LOX-1, as well as tubular immunoreactivity of inducible nitric oxide synthase, nitrotyrosine, nuclear factor-κB, transforming growth factor-β, and tubulointerstitial and perivascular trichrome staining.ConclusionET-A blockade improves renal hemodynamic and function in HC, and decreases oxidative stress, and renal vascular and tubulointerstitial inflammation and fibrosis. These findings support a role for the endogenous ET system in renal injury in HC and atherosclerosis

Oxidized Low-Density Lipoproteins Induce Tissue Factor Expression in T-Lymphocytes via activation of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1

T-lymphocytes plays an important role in the pathophysiology of acute coronary syndromes (ACS). T-cell activation in vitro by pro-inflammatory cytokines may lead to functional Tissue Factor (TF) expression, indicating a possible contribution of immunity to thrombosis. Oxidized low-density lipoproteins (oxLDLs) are found abundantly in atherosclerotic plaques. We aimed at evaluating the effects of oxLDLs on TF expression in T-cells and the role of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)

Standardizing Scavenger Receptor Nomenclature

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a variety of ligands, including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the U.S. National Institute of Allergy and Infectious Diseases, National Institutes of Health to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of non-self or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. The discussion and nomenclature recommendations described in this report only refer to mammalian scavenger receptors. The purpose of this article is to describe the proposed mammalian nomenclature and classification developed at the workshop and to solicit additional feedback from the broader research community

“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 oxidised low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified 5 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-labelled oxLDL by the LOX-1-expressing cell model was inhibited with sub-nanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p<0.01) by pre-incubation 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