"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.

LDL uptake-dependent phosphatidylethanolamine translocation to the cell surface promotes fusion of osteoclast-like cells.

Osteoporosis is associated with vessel diseases attributed to hyperlipidemia, and bone resorption by multinucleated osteoclasts is related to lipid metabolism. In this study, we generated low-density lipoprotein receptor (LDLR)/lectin-like oxidized LDL receptor-1 (LOX-1, also known as Olr1) double knockout (dKO) mice. We found that, like LDLR single KO (sKO), LDLR/LOX-1 dKO impaired cell-cell fusion of osteoclast-like cells (OCLs). LDLR/LOX-1 dKO and LDLR sKO preosteoclasts exhibited decreased uptake of LDL. The cell surface cholesterol levels of both LDLR/LOX-1 dKO and LDLR sKO osteoclasts were lower than the levels of wild-type OCLs. Additionally, the amount of phosphatidylethanolamine (PE) on the cell surface was attenuated in LDLR/LOX-1 dKO and LDLR sKO preosteoclasts, whereas the PE distribution in wild-type OCLs was concentrated on the filopodia in contact with neighboring cells. Abrogation of the ATP binding cassette G1 (ABCG1) transporter, which transfers PE to the cell surface, caused decreased PE translocation to the cell surface and subsequent cell-cell fusion. The findings of this study indicate the involvement of a novel cascade (LDLR∼ABCG1∼PE translocation to cell surface∼cell-cell fusion) in multinucleation of OCLs.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1): a crucial driver of atherosclerotic cardiovascular disease.

Cardiovascular diseases (CVDs), specifically lipid-driven atherosclerotic CVDs, remain the number one cause of death worldwide. The lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1), a scavenger receptor that promotes endothelial dysfunction by inducing pro-atherogenic signalling and plaque formation via the endothelial uptake of oxidized LDL (oxLDL) and electronegative LDL, contributes to the initiation, progression, and destabilization of atheromatous plaques, eventually leading to the development of myocardial infarction and certain forms of stroke. In addition to its expression in endothelial cells, LOX-1 is expressed in macrophages, cardiomyocytes, fibroblasts, dendritic cells, lymphocytes, and neutrophils, further implicating this receptor in multiple aspects of atherosclerotic plaque formation. LOX-1 holds promise as a novel diagnostic and therapeutic target for certain CVDs; therefore, understanding the molecular structure and function of LOX-1 is of critical importance. In this review, we highlight the latest scientific findings related to LOX-1, its ligands, and their roles in the broad spectrum of CVDs. We describe recent findings from basic research, delineate their translational value, and discuss the potential of LOX-1 as a novel target for the prevention, diagnosis, and treatment of related CVDs.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1): a crucial driver of atherosclerotic cardiovascular disease.

Cardiovascular diseases (CVDs), specifically lipid-driven atherosclerotic CVDs, remain the number one cause of death worldwide. The lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1), a scavenger receptor that promotes endothelial dysfunction by inducing pro-atherogenic signalling and plaque formation via the endothelial uptake of oxidized LDL (oxLDL) and electronegative LDL, contributes to the initiation, progression, and destabilization of atheromatous plaques, eventually leading to the development of myocardial infarction and certain forms of stroke. In addition to its expression in endothelial cells, LOX-1 is expressed in macrophages, cardiomyocytes, fibroblasts, dendritic cells, lymphocytes, and neutrophils, further implicating this receptor in multiple aspects of atherosclerotic plaque formation. LOX-1 holds promise as a novel diagnostic and therapeutic target for certain CVDs; therefore, understanding the molecular structure and function of LOX-1 is of critical importance. In this review, we highlight the latest scientific findings related to LOX-1, its ligands, and their roles in the broad spectrum of CVDs. We describe recent findings from basic research, delineate their translational value, and discuss the potential of LOX-1 as a novel target for the prevention, diagnosis, and treatment of related CVDs.

Adiponectin forms a complex with atherogenic LDL and inhibits its downstream effects.

Adiponectin, an adipocyte-derived protein, has antiatherogenic and antidiabetic effects, but how it confers the atherogenic effects is not well known. To study the antiatherogenic mechanisms of adiponectin, we examined whether it interacts with atherogenic low density lipoprotein (LDL) to attenuate LDL's atherogenicity. L5, the most electronegative subfraction of LDL, induces atherogenic responses similarly to copper-oxidized LDL (oxLDL). Unlike the native LDL endocytosed via the LDL receptor, L5 and oxLDL are internalized by cells via the lectin-like oxidized LDL receptor-1 (LOX-1). Using enzyme-linked immunosorbent assays (ELISAs), we showed that adiponectin preferentially bound oxLDL but not native LDL. In Chinese hamster ovary (CHO) cells transfected with the LOX-1 or LDL receptor, adiponectin selectively inhibited the uptake of oxLDL but not of native LDL, respectively. Furthermore, adiponectin suppressed the internalization of oxLDL in human coronary artery endothelial cells (HCAECs) and THP-1-derived macrophages. Western blot analysis of human plasma showed that adiponectin was abundant in L5 but not in L1, the least electronegative subfraction of LDL. Sandwich ELISAs with anti-adiponectin and anti-apolipoprotein B antibodies confirmed the binding of adiponectin to L5 and oxLDL. In LOX-1-expressing CHO cells, adiponectin inhibited cellular responses to oxLDL and L5, including nuclear factor-κB activation and extracellular signal-regulated kinas phosphorylation. In HCAECs, adiponectin inhibited oxLDL-induced endothelin-1 secretion and extracellular signal-regulated kinase phosphorylation. Conversely, oxLDL suppressed the adiponectin-induced activation of adenosine monophosphate-activated protein kinase in COS-7 cells expressing adiponectin receptor AdipoR1. Our findings suggest that adiponectin binds and inactivates atherogenic LDL, providing novel insight into the antiatherogenic mechanisms of adiponectin.

Quantitative and qualitative lipid improvement with chronic hepatitis C virus eradication using direct-acting antivirals.

AIM: Direct-acting antivirals have revolutionized hepatitis C virus (HCV) therapy by providing a high sustained virological response (SVR) rate and subsequent favorable lipid increases. Proprotein convertase subtilisin-kexin like-9 (PCSK9) plays an important role in regulating quantitative lipid levels. This study examined the interactions between quantitative PCSK9 and lipid changes, as well as qualitative lipid changes in terms of lectin-like oxidized low-density lipoprotein (LDL) receptor-1 ligand containing apolipoprotein B (LAB) and high-density lipoprotein (HDL) cholesterol uptake capacity (HDL-CUC). METHODS: Patients with chronic HCV infection (N = 231) who achieved an SVR by direct-acting antivirals without lipid-lowering therapy were included for comparisons of PCSK9, LAB, HDL-CUC, and other clinical indices between pretreatment and SVR12 time points. RESULTS: LDL (LDL) cholesterol and HDL cholesterol levels were quantitatively increased at SVR12, along with higher PCSK9 (all p < 0.0001). PCSK9 was significantly correlated with LDL cholesterol (r = 0.244, p = 0.0003) and apolipoprotein B (r = 0.222, p = 0.0009) at SVR12. Regarding qualitative LDL changes, LAB was significantly decreased and LAB/LDL cholesterol and LAB/apolipoprotein B proportions were improved at SVR12 (all p < 0.0001). In terms of qualitative HDL changes, HDL-CUC was significantly ameliorated, along with HDL-CUC/HDL cholesterol, HDL-CUC/ apolipoprotein A1, and HDL-CUC/ apolipoprotein A2 at SVR12 (all p < 0.0001). CONCLUSIONS: HCV eradication by direct-acting antivirals may produce quantitative lipid profile changes, along with PCSK9 production recovery in addition to qualitative lipid improvement, which possibly confers the additional secondary benefits of atherosclerosis improvement and cardiovascular disease event reduction.

LOX-1 (Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1) Deletion Has Protective Effects on Stroke in the Genetic Background of Stroke-Prone Spontaneously Hypertensive Rat.

Background and Purpose- oxLDL (oxidized low-density lipoprotein) has been known for its potential to induce endothelial dysfunction and used as a major serological marker of oxidative stress. Recently, LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a lectin-like receptor for oxLDL, has attracted attention in studies of neuronal apoptosis and stroke. We aim to investigate the impact of LOX-1-deficiency on spontaneous hypertension-related brain damage in the present study. Methods- We generated a LOX-1 deficient strain on the genetic background of stroke-prone spontaneously hypertensive rat (SHRSP), an animal model of severe hypertension and spontaneous stroke. In this new disease model with stroke-proneness, we monitored the occurrence of brain abnormalities with and without salt loading by multiple procedures including T2 weighted magnetic resonance imaging and also explored circulatory miRNAs as diagnostic biomarkers for cerebral ischemic injury by microarray analysis. Results- Both T2 weighted magnetic resonance imaging abnormalities and physiological parameter changes could be detected at significantly delayed timing in LOX-1 knockout rats compared with wild-type SHRSP, in either case of normal rat chow and salt loading (P<0.005 in all instances; n=11-20 for SHRSP and n=13-23 for LOX-1 knockout rats). There were no significant differences in the form of magnetic resonance imaging findings between the strains. A number of miRNAs expressed in the normal rat plasma, including rno-miR-150-5p and rno-miR-320-3p, showed significant changes after spontaneous brain damage in SHRSP, whereas the corresponding changes were modest or almost unnoticeable in LOX-1 knockout rats. There appeared to be the lessening of correlation of postischemic miRNA alterations between the injured brain tissue and plasma in LOX-1 knockout rats. Conclusions- Our data show that deficiency of LOX-1 has a protective effect on spontaneous brain damage in a newly generated LOX-1-deficient strain of SHRSP. Further, our analysis of miRNAs as biomarkers for ischemic brain damage supports a potential involvement of LOX-1 in blood brain barrier disruption after cerebral ischemia. Visual Overview- An online visual overview is available for this article.

High-cholesterol diet during pregnancy induces maternal vascular dysfunction in mice: potential role for oxidized LDL-induced LOX-1 and AT1 receptor activation.

The lectin-like oxidized low-density-lipoprotein (oxLDL) receptor-1 (LOX-1) has been shown to induce angiotensin II (AngII) type 1 receptor (AT1) activation, contributing to vascular dysfunction. Preeclampsia is a pregnancy complication characterized by vascular dysfunction and increased LOX-1 and AT1 activation; however, whether LOX-1 and AT1 activity contributes to vascular dysfunction in preeclampsia is unknown. We hypothesized that increased oxLDL levels during pregnancy lead to LOX-1 activation and subsequent AT1 activation, resulting in vascular dysfunction. Pregnant wild-type (WT) and transgenic LOX-1 overexpressing (LOX-1tg) mice were fed a control diet (CD) or high-cholesterol diet (HCD, to impair vascular function) between gestational day (GD) 13.5-GD18.5. On GD18.5, AngII-induced vasoconstriction and methylcholine (MCh)-induced endothelium-dependent vasodilation responses were assessed in aortas and uterine arteries. HCD decreased fetal weight and increased circulating oxLDL/cholesterol levels in WT, but not in LOX-1tg mice. HCD did not alter AngII responsiveness or AT1 expression in both vascular beds; however, AngII responsiveness and AT1 expression were lower in aortas from LOX-1tg compared with WT mice. In aortas from WT-CD mice, acute oxLDL exposure induced AT1-mediated vasoconstriction via LOX-1. HCD impaired endothelium-dependent vasodilation and increased superoxide levels in WT aortas, but not uterine arteries. Moreover, in WT-CD mice oxLDL decreased MCh sensitivity in both vascular beds, partially via LOX-1. In summary, HCD impaired pregnancy outcomes and vascular function, and oxLDL-induced LOX-1 activation may contribute to vascular dysfunction via AT1. Our study suggests that LOX-1 could be a potential target to prevent adverse outcomes associated with vascular dysfunction in preeclampsia.

Atherogenic L5 LDL induces cardiomyocyte apoptosis and inhibits KATP channels through CaMKII activation.

BACKGROUND: Cardiac Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation plays a critical role in cardiomyocyte (CM) apoptosis and arrhythmia. Functional ATP-sensitive potassium (KATP) channels are essential for cardiac protection during ischemia. In cultured CMs, L5 low-density lipoprotein (LDL) induces apoptosis and QTc prolongation. L5 is a highly electronegative and atherogenic aberrant form of LDL, and its levels are significantly higher in patients with cardiovascular-related diseases. Here, the role of L5 in cardiac injury was studied by evaluating the effects of L5 on CaMKII activity and KATP channel physiology in CMs. METHODS: Cultured neonatal rat CMs (NRCMs) were treated with a moderate concentration (ie, 7.5 µg/mL) of L5 or L1 (the least electronegative LDL subfraction). NRCMs were examined for apoptosis and viability, CaMKII activity, and the expression of phosphorylated CaMKIIδ and NOX2/gp91phox. The function of KATP and action potentials (APs) was analyzed by using the patch-clamp technique. RESULTS: In NRCMs, L5 but not L1 significantly induced cell apoptosis and reduced cell viability. Furthermore, L5 decreased Kir6.2 expression by more than 50%. Patch-clamp analysis showed that L5 reduced the KATP current (IKATP) density induced by pinacidil, a KATP opener. The partial recovery of the inward potassium current during pinacidil washout was susceptible to subsequent inhibition by the IKATP blocker glibenclamide. Suppression of IKATP by L5 significantly prolonged the AP duration. L5 also significantly increased the activity of CaMKII, the phosphorylation of CaMKIIδ, and the expression of NOX2/gp91phox. L5-induced apoptosis was prevented by the addition of the CaMKII inhibitor KN93 and the reactive oxygen species scavenger Mn (III)TBAP. CONCLUSIONS: L5 but not L1 induces CM damage through the activation of the CaMKII pathway and increases arrhythmogenicity in CMs by modulating the AP duration. These results help to explain the harmful effects of L5 in cardiovascular-related disease.

A Consensus Definitive Classification of Scavenger Receptors and Their Roles in Health and Disease.

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 diverse 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 United States 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 nonself 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. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.

Plasma L5 levels are elevated in ischemic stroke patients and enhance platelet aggregation.

L5, the most electronegative and atherogenic subfraction of low-density lipoprotein (LDL), induces platelet activation. We hypothesized that plasma L5 levels are increased in acute ischemic stroke patients and examined whether lectin-like oxidized LDL receptor-1 (LOX-1), the receptor for L5 on endothelial cells and platelets, plays a critical role in stroke. Because amyloid ß (Aß) stimulates platelet aggregation, we studied whether L5 and Aß function synergistically to induce prothrombotic pathways leading to stroke. Levels of plasma L5, serum Aß, and platelet LOX-1 expression were significantly higher in acute ischemic stroke patients than in controls without metabolic syndrome (P < .01). In mice subjected to focal cerebral ischemia, L5 treatment resulted in larger infarction volumes than did phosphate-buffered saline treatment. Deficiency or neutralizing of LOX-1 reduced infarct volume up to threefold after focal cerebral ischemia in mice, illustrating the importance of LOX-1 in stroke injury. In human platelets, L5 but not L1 (the least electronegative LDL subfraction) induced Aß release via IκB kinase 2 (IKK2). Furthermore, L5+Aß synergistically induced glycoprotein IIb/IIIa receptor activation; phosphorylation of IKK2, IκBα, p65, and c-Jun N-terminal kinase 1; and platelet aggregation. These effects were blocked by inhibiting IKK2, LOX-1, or nuclear factor-κB (NF-κB). Injecting L5+Aß shortened tail-bleeding time by 50% (n = 12; P < .05 vs L1-injected mice), which was prevented by the IKK2 inhibitor. Our findings suggest that, through LOX-1, atherogenic L5 potentiates Aß-mediated platelet activation, platelet aggregation, and hemostasis via IKK2/NF-κB signaling. L5 elevation may be a risk factor for cerebral atherothrombosis, and downregulating LOX-1 and inhibiting IKK2 may be novel antithrombotic strategies.

Alterations in vascular function by syncytiotrophoblast extracellular vesicles via lectin-like oxidized low-density lipoprotein receptor-1 in mouse uterine arteries.

Syncytiotrophoblast extracellular vesicles (STBEVs), released into the maternal circulation during pregnancy, have been shown to affect vascular function; however, the mechanism remains unknown. In rats, STBEVs were shown to reduce endothelium-mediated vasodilation via lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a multi-ligand scavenger receptor that has been associated with vascular dysfunction. Recently, LOX-1 was shown to interact with the angiotensin II type 1 receptor (AT-1). We hypothesized that, in pregnant mice, STBEVs would impair vascular function via LOX-1 and would specifically affect angiotensin II responses. Uterine arteries from pregnant control (C57BL/6) and LOX-1 knockout (LOX-1KO) mice were isolated on gestational day (GD) 18.5. Endothelium-dependent (methylcholine (MCh); ± N(G)-Nitro-L-arginine methyl ester to assess nitric oxide (NO) contribution), and -independent (sodium nitroprusside) vasodilation, and vasoconstriction (angiotensin II; ± AT-1 [candesartan] or angiotensin II type 2 receptor (AT-2) [PD123.319] receptor antagonists; high potassium salt solution) responses were assessed using wire myography. AT-1 and AT-2 expression was analyzed using fluorescence microscopy. Human umbilical vein endothelial cells (HUVECs) were stimulated with STBEVs ± LOX-1 blocking antibody, and superoxide and peroxynitrite production were analyzed. Although MCh-induced vasodilation was decreased (P=0.0012), NO contribution to vasodilation was greater in LOX-1KO mice (P=0.0055). STBEVs delayed angiotensin II tachyphylaxis in arteries from control but not LOX-1KO mice (P<0.0001), while AT-1 and AT-2 expression was unchanged. STBEVs increased peroxynitrite production in HUVECs via LOX-1 (P=0.0091). In summary, LOX-1 deletion altered endothelium-mediated vasodilation, suggesting that LOX-1 contributes to vascular adaptations in pregnancy. STBEVs increased angiotensin II responsiveness and oxidative stress levels via LOX-1, suggesting that increased LOX-1 expression/activation or STBEVs could adversely affect vascular function and contribute to vascular complications of pregnancy.

Smoking cessation reduces the lectin-like low-density lipoprotein receptor index, an independent cardiovascular risk marker of vascular inflammation.

Vessel wall inflammation promotes the destabilization of atherosclerotic plaques. The lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) expressed by vascular cells and monocytes. LOX index is calculated by multiplying LOX-1 ligand containing apolipoprotein B level with the soluble LOX-1. A high LOX index reflects an increased risk for stroke and myocardial infarction. However, the change in LOX index after smoking cessation and the relationship between smoking-related variables and LOX index are unknown. Relation of the clinical parameters to the LOX index was examined on 180 subjects (135 males and 45 females) at the first visit to our outpatient clinic for smoking cessation. The impact of smoking cessation on the LOX index was also determined in the 94 subjects (62 males and 32 females) who successfully stopped smoking. Sex-adjusted regression analysis and multivariate analysis identified three independent determinants of the LOX index, namely, low-density lipoprotein-cholesterol (LDL-C; ß = 0.311, p < 0.001), high-sensitivity C-reactive protein (ß = 0.358, p < 0.001), and expired carbon monoxide concentration reflecting smoking heaviness (ß = 0.264, p = 0.003). Body mass index (BMI) significantly increased 3 months after the onset of smoking cessation (p < 0.001). However, the LOX index significantly decreased (p < 0.001), regardless of the rate of increase in BMI post-cessation. The LOX index is closely associated with smoking heaviness as well as dyslipidemia and an inflammation marker. Smoking cessation may induce a decrease in this cardiovascular risk marker, independently of weight gain.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) in sickle cell disease vasculopathy.

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL. Increased expression of LOX-1 has been demonstrated in atherosclerotic lesions and diabetic vasculopathy. In this study, we investigate the expression of LOX-1 receptor in sickle cell disease (SCD) vasculopathy. Expression of LOX-1 in brain vascular endothelium is markedly increased and LOX-1 gene expression is upregulated in cultured human brain microvascular endothelial cells by incubation with SCD erythrocytes. Also, the level of circulating soluble LOX-1 concentration is elevated in the plasma of SCD patients. Increased LOX-1 expression in endothelial cells is potentially involved in the pathogenesis of SCD vasculopathy. Soluble LOX-1 concentration in SCD may provide a novel biomarker for risk stratification of sickle cell vascular complications.

Interplay between CRP, Atherogenic LDL, and LOX-1 and Its Potential Role in the Pathogenesis of Atherosclerosis.

BACKGROUND: Studies have shown that the classic acute-phase protein C-reactive protein (CRP) has proinflammatory effects on vascular cells and may play a causal role in the pathogenesis of coronary artery disease. A growing body of evidence has suggested that interplay between CRP, lectin-like oxidized LDL receptor-1 (LOX-1), and atherogenic LDL may underlie the mechanism of endothelial dysfunction that leads to atherosclerosis. CONTENT: We review the biochemical evidence for an association of CRP, LOX-1, and either oxidized LDL (OxLDL) or electronegative L5 LDL with the pathogenesis of coronary artery disease. Artificially oxidized OxLDL has been studied extensively for its role in atherogenesis, as has electronegative L5 LDL, which is present at increased levels in patients with increased cardiovascular risks. OxLDL and L5 have been shown to stimulate human aortic endothelial cells to produce CRP, indicating that CRP is synthesized locally in the endothelium. The ligand-binding face (B-face) of CRP has been shown to bind the LOX-1 scavenger receptor and increase LOX-1 expression in endothelial cells, thereby promoting the uptake of OxLDL or L5 by LOX-1 into endothelial cells to induce endothelial dysfunction. SUMMARY: CRP and LOX-1 may form a positive feedback loop with OxLDL or L5 in atherogenesis, whereby increased levels of atherogenic LDL in patients with cardiovascular risks induce endothelial cells to express CRP, which may in turn increase the expression of LOX-1 to promote the uptake of atherogenic LDL into endothelial cells. Further research is needed to confirm a causal role for CRP in atherogenesis.

Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor.

The angiotensin II type 1 receptor (AT1) is a 7-transmembrane domain GPCR that when activated by its ligand angiotensin II, generates signaling events promoting vascular dysfunction and the development of cardiovascular disease. Here, we show that the single-transmembrane oxidized LDL (oxLDL) receptor (LOX-1) resides in proximity to AT1 on cell-surface membranes and that binding of oxLDL to LOX-1 can allosterically activate AT1-dependent signaling events. oxLDL-induced signaling events in human vascular endothelial cells were abolished by knockdown of AT1 and inhibited by AT1 blockade (ARB). oxLDL increased cytosolic G protein by 350% in Chinese hamster ovary (CHO) cells with genetically induced expression of AT1 and LOX-1, whereas little increase was observed in CHO cells expressing only LOX-1. Immunoprecipitation and in situ proximity ligation assay (PLA) assays in CHO cells revealed the presence of cell-surface complexes involving LOX-1 and AT1. Chimeric analysis showed that oxLDL-induced AT1 signaling events are mediated via interactions between the intracellular domain of LOX-1 and AT1 that activate AT1. oxLDL-induced impairment of endothelium-dependent vascular relaxation of vascular ring from mouse thoracic aorta was abolished by ARB or genetic deletion of AT1. These findings reveal a novel pathway for AT1 activation and suggest a new mechanism whereby oxLDL may be promoting risk for cardiovascular disease.

Developmental Endothelial Locus-1 (Del-1) Inhibits Oxidized Low-Density Lipoprotein Activity by Direct Binding, and Its Overexpression Attenuates Atherogenesis in Mice.

BACKGROUND: Modified low-density lipoprotein (LDL) binding to scavenger receptors has been implicated in atherosclerosis. It is hypothesized that a third molecule may affect modified LDL binding, therefore, this study focuses on the soluble endogenous protein, developmental endothelial locus-1 (Del-1), as an inhibitor of oxidized LDL (oxLDL) interactions.Methods and Results:Del-1 preferentially bound oxLDL over native LDL in a cell-free binding assay. Del-1 also inhibited DiI-labeled oxLDL uptake by scavenger receptors irrespective of the receptor type (LOX-1, SR-AI, CD36, or SR-BI) expressed in COS-7 cells, and independent of cell type (human coronary artery endothelial cells (HCAECs) or THP-1-derived macrophages). Furthermore, Del-1 suppressed oxLDL-inducedMCP-1andICAM-1expression and endothelin-1 secretion in HCAECs. Then, male Del-1 transgenic (Del-1Tg) and wild-type mice (WT) mice were established and fed a Paigen diet for 20 weeks from the age of 24 weeks. While plasma lipid concentrations did not differ between WT and Del-1Tg mice, plasma LOX-1-ligand activity was significantly lower in Del-1Tg than in WT mice. Moreover, lipid accumulation in aortic roots was significantly less in the Del-1Tg mice, evaluated with Oil red-O. Taken together, Del-1 appears to block the activity of oxLDL pharmacologically by direct binding in vitro, and attenuates atherogenesis in vivo, although its role in physiological settings are yet to be resolved. CONCLUSIONS: Del-1 intercepted oxLDL before its receptor binding to reduce atherogenesis. (Circ J 2016; 80: 2541-2549).

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.

Increased LDL electronegativity in chronic kidney disease disrupts calcium homeostasis resulting in cardiac dysfunction.

Chronic kidney disease (CKD), an independent risk factor for cardiovascular disease, is associated with abnormal lipoprotein metabolism. We examined whether electronegative low-density lipoprotein (LDL) is mechanistically linked to cardiac dysfunction in patients with early CKD. We compared echocardiographic parameters between patients with stage 2 CKD (n = 88) and normal controls (n = 89) and found that impaired relaxation was more common in CKD patients. Reduction in estimated glomerular filtration rate was an independent predictor of left ventricular relaxation dysfunction. We then examined cardiac function in a rat model of early CKD induced by unilateral nephrectomy (UNx) by analyzing pressure-volume loop data. The time constant of isovolumic pressure decay was longer and the maximal velocity of pressure fall was slower in UNx rats than in controls. When we investigated the mechanisms underlying relaxation dysfunction, we found that LDL from CKD patients and UNx rats was more electronegative than LDL from their respective controls and that LDL from UNx rats induced intracellular calcium overload in H9c2 cardiomyocytes in vitro. Furthermore, chronic administration of electronegative LDL, which signals through lectin-like oxidized LDL receptor-1 (LOX-1), induced relaxation dysfunction in wild-type but not LOX-1(-/-) mice. In in vitro and in vivo experiments, impaired cardiac relaxation was associated with increased calcium transient resulting from nitric oxide (NO)-dependent nitrosylation of SERCA2a due to increases in inducible NO synthase expression and endothelial NO synthase uncoupling. In conclusion, LDL becomes more electronegative in early CKD. This change disrupts SERCA2a-regulated calcium homeostasis, which may be the mechanism underlying cardiorenal syndrome.

Effect of sodium tanshinone IIA sulfonate treatment in a rat model of preeclampsia.

Preeclampsia is a disorder of pregnancy with a significant impact on maternal and fetal health. The complexity of this multifactorial condition has precluded development of effective therapies and, although many potential pathways have been investigated, the etiology still requires clarification. Our group has investigated the scavenger lectin-like oxidized LDL (LOX-1) receptor, which may respond to factors released from the distressed placenta that contribute to the vascular pathologies observed in preeclampsia. Given the known beneficial effects of sodium tanshinone IIA sulfonate (STS; a component of Salvia miltiorrhiza) on vasodilation, reduction of oxidative stress, and lipid profiles, we have investigated its role as a potential treatment strategy. We hypothesized that STS would improve vascular endothelial function and, combined with a reduction in oxidative stress, would improve pregnancy outcomes in a rat model of preeclampsia (reduced uteroplacental perfusion pressure, RUPP). We further hypothesized this may occur via the action of STS on the LOX-1 and/or platelet-activating factor (PAF) receptor axes. The RUPP model increased maternal blood pressure, vascular oxidative stress, and involvement of the vascular PAF receptor. Treatment with STS during pregnancy decreased both oxidative stress and involvement of the PAF receptor; however, it also increased involvement of the LOX-1 receptor, which is in line with the concept that scavenger receptors, such as LOX-1 and PAF, are upregulated in response to ligand binding and/or under pathological conditions. In this model of preeclampsia, however, the vascular actions of STS did not lead to improvements in pregnancy outcome such as fetal biometrics or maternal blood pressure.