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RATIONALE - Atherosclerosis is, in part, caused by immune and inflammatory cell infiltration into the vascular wall, leading to enhanced inflammation and lipid accumulation in the aortic endothelium. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are critical regulators of atherogenicity. Given the fundamental contribution lesion macrophages make to fuel atherosclerosis, whether and how myeloid-specific epsins promote atherogenesis is an open and significant question.
OBJECTIVE - We will determine the role of myeloid-specific epsins in regulating lesion macrophage function during atherosclerosis.
METHODS AND RESULTS - We engineered myeloid cell-specific epsins double knockout mice (LysM-DKO) on an ApoE background. On Western diet, these mice exhibited marked decrease in atherosclerotic lesion formation, diminished immune and inflammatory cell content in aortas, and reduced necrotic core content but increased smooth muscle cell content in aortic root sections. Epsins deficiency hindered foam cell formation and suppressed proinflammatory macrophage phenotype but increased efferocytosis and anti-inflammatory macrophage phenotype in primary macrophages. Mechanistically, we show that epsin loss specifically increased total and surface levels of LRP-1 (LDLR [low-density lipoprotein receptor]-related protein 1), an efferocytosis receptor with antiatherosclerotic properties. We further show that epsin and LRP-1 interact via epsin's ubiquitin-interacting motif domain. ox-LDL (oxidized LDL) treatment increased LRP-1 ubiquitination, subsequent binding to epsin, and its internalization from the cell surface, suggesting that epsins promote the ubiquitin-dependent internalization and downregulation of LRP-1. Crossing ApoE/LysM-DKO mice onto an LRP-1 heterozygous background restored, in part, atherosclerosis, suggesting that epsin-mediated LRP-1 downregulation in macrophages plays a pivotal role in propelling atherogenesis.
CONCLUSIONS - Myeloid epsins promote atherogenesis by facilitating proinflammatory macrophage recruitment and inhibiting efferocytosis in part by downregulating LRP-1, implicating that targeting epsins in macrophages may serve as a novel therapeutic strategy to treat atherosclerosis.
BACKGROUND - We previously showed that mice lacking MΦLRP1 (low-density lipoprotein receptor-related protein 1 in macrophages) undergo accelerated atherosclerotic plaque formation due to changes in macrophages including increased apoptosis, decreased efferocytosis, and exaggerated transition to the inflammatory M1 phenotype. Here we sought to explore the role of macrophage low-density lipoprotein receptor-related protein 1 during regression of atherosclerosis since regressing plaques are characterized by transitioning of macrophages to M2 status as inflammation resolves.
METHODS - Apolipoprotein E mice on a high-fat diet for 12 weeks were reconstituted with bone marrow from apolipoprotein E-producing wild-type or MΦLRP1 mice, and then placed on a chow diet for 10 weeks (n=9 to 11 mice/group). A cohort of apolipoprotein E mice reconstituted with apolipoprotein E bone marrow served as baseline controls (n=9).
RESULTS - Plaques of both wild-type and MΦLRP1 bone marrow recipients regressed compared with controls (11% and 22%, respectively; P<0.05), and plaques of MΦLRP1 recipients were 13% smaller than those of wild-type recipients ( P<0.05). Recipients of MΦLRP1 marrow had 36% fewer M1 macrophages ( P<0.01) and 2.5-fold more CCR7 (C-C chemokine receptor type 7)-positive macrophages in the plaque relative to wild-type mice ( P<0.01). Additionally, in vivo studies of cellular egress showed a 4.6-fold increase in 5-ethynyl-2´-deoxyuridine-labeled CCR7 macrophages in mediastinal lymph nodes. Finally, in vivo studies of reverse cholesterol transport showed a 1.4-fold higher reverse cholesterol transport in MΦLRP1 recipient mice ( P<0.01).
CONCLUSIONS - Absence of macrophage low-density lipoprotein receptor-related protein 1 unexpectedly accelerates atherosclerosis regression, enhances reverse cholesterol transport, and increases expression of the motility receptor CCR7, which drives macrophage egress from lesions.
OBJECTIVE - Antiatherosclerotic effects of tumor necrosis factor-α (TNF-α) blockade in patients with systemic inflammatory states are not conclusively demonstrated, which suggests that effects depend on the cause of inflammation. Macrophage LRP1 (low-density lipoprotein receptor-related protein 1) and apoE contribute to inflammation through different pathways. We studied the antiatherosclerosis effects of TNF-α blockade in hyperlipidemic mice lacking either LRP1 (MΦLRP1(-/-)) or apoE from macrophages.
APPROACH AND RESULTS - Lethally irradiated low-density lipoprotein receptor (LDLR)(-/-) mice were reconstituted with bone marrow from either wild-type, MΦLRP1(-/-), apoE(-/-) or apoE(-/-)/MΦLRP1(-/-)(DKO) mice, and then treated with the TNF-α inhibitor adalimumab while fed a Western-type diet. Adalimumab reduced plasma TNF-α concentration, suppressed blood ly6C(hi) monocyte levels and their migration into the lesion, and reduced lesion cellularity and inflammation in both wild-type→LDLR(-/-) and apoE(-/-)→LDLR(-/-) mice. Overall, adalimumab reduced lesion burden by 52% to 57% in these mice. Adalimumab reduced TNF-α and blood ly6C(hi) monocyte levels in MΦLRP1(-/-)→LDLR(-/-) and DKO→LDLR(-/-) mice, but it did not suppress ly6C(hi) monocyte migration into the lesion or atherosclerosis progression.
CONCLUSIONS - Our results show that TNF-α blockade exerts antiatherosclerotic effects that are dependent on the presence of macrophage LRP1.
© 2016 American Heart Association, Inc.
Expression of molecules involved in lipid homeostasis such as the low density lipoprotein receptor (LDLr) on antigen presenting cells (APCs) has been shown to enhance invariant natural killer T (iNKT) cell function. However, the contribution to iNKT cell activation by other lipoprotein receptors with shared structural and ligand binding properties to the LDLr has not been described. In this study, we investigated whether a structurally related receptor to the LDLr, known as LDL receptor-related protein (LRP), plays a role in iNKT cell activation. We found that, unlike the LDLr which is highly expressed on all immune cells, the LRP was preferentially expressed at high levels on F4/80+ macrophages (MΦ). We also show that CD169+ MΦs, known to present antigen to iNKT cells, exhibited increased expression of LRP compared to CD169- MΦs. To test the contribution of MΦ LRP to iNKT cell activation we used a mouse model of MΦ LRP conditional knockout (LRP-cKO). LRP-cKO MΦs pulsed with glycolipid alpha-galactosylceramide (αGC) elicited normal IL-2 secretion by iNKT hybridoma and in vivo challenge of LRP-cKO mice led to normal IFN-γ, but blunted IL-4 response in both serum and intracellular expression by iNKT cells. Flow cytometric analyses show similar levels of MHC class-I like molecule CD1d on LRP-cKO MΦs and normal glycolipid uptake. Survey of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages and no homeostatic disruption as evidenced by the absence of programmed death-1 and Ly-49 surface receptors. Mixed bone marrow chimeras showed that the inability iNKT cells to make IL-4 is cell extrinsic and can be rescued in the presence of wild type APCs. Collectively, these data demonstrate that, although MΦ LRP may not be necessary for IFN-γ responses, it can contribute to iNKT cell activation by enhancing early IL-4 secretion.
Sepp1 supplies selenium to tissues via receptor-mediated endocytosis. Mice, rats, and humans have 10 selenocysteines in Sepp1, which are incorporated via recoding of the stop codon, UGA. Four isoforms of rat Sepp1 have been identified, including full-length Sepp1 and three others, which terminate at the second, third, and seventh UGA codons. Previous studies have shown that the longer Sepp1 isoforms bind to the low density lipoprotein receptor apoER2, but the mechanism remains unclear. To identify the essential residues for apoER2 binding, an in vitro Sepp1 binding assay was developed using different Sec to Cys substituted variants of Sepp1 produced in HEK293T cells. ApoER2 was found to bind the two longest isoforms. These results suggest that Sepp1 isoforms with six or more selenocysteines are taken up by apoER2. Furthermore, the C-terminal domain of Sepp1 alone can bind to apoER2. These results indicate that apoER2 binds to the Sepp1 C-terminal domain and does not require the heparin-binding site, which is located in the N-terminal domain. Site-directed mutagenesis identified three residues of Sepp1 that are necessary for apoER2 binding. Sequential deletion of extracellular domains of apoER2 surprisingly identified the YWTD β-propeller domain as the Sepp1 binding site. Finally, we show that apoER2 missing the ligand-binding repeat region, which can result from cleavage at a furin cleavage site present in some apoER2 isoforms, can act as a receptor for Sepp1. Thus, longer isoforms of Sepp1 with high selenium content interact with a binding site distinct from the ligand-binding domain of apoER2 for selenium delivery.
BACKGROUND AND AIMS - In vitro studies suggest that low density lipoprotein receptor-related protein 1 (LRP1) plays a role in the secondary uptake of chylomicrons. In addition, in vivo studies using LRP-1 knockout mice show these animals exhibit delayed chylomicron clearance. Whether this is true in humans is unknown. We aimed to determine whether genetic variants in LRP-1 are associated with postprandial chylomicron uptake in humans given an oral fat challenge.
METHODS AND RESULTS - As many as 817 men and women (mean age +/- standard deviation = 48.4 +/- 16.4 years) forming the study population for the Genetics of Lipid Lowering Drugs Network (GOLDN) study ingested an oral fat load of 700 kilocalories per m² of body surface area at 83% fat, after an 8-h fast. Chylomicrons were measured by nuclear resonance spectroscopy (NMR) at fasting, and 3.5 and 6 h after the meal. 26 Single nucleotide polymorphisms (SNPs) in the LRP-1 gene were genotyped on the Affymetrix 6.0 array. Chylomicrons were, as expected, zero at fasting. Mixed linear models adjusted for age, sex, study site and pedigree tested for associations between LRP-1 SNPs and changes in chylomicron concentrations 3.5-6 h. A gene-based test across all 26 SNPs was conducted which corrected for the linkage disequilibrium (LD) between SNPs. 11 LRP-1 SNPs were significantly associated with the change in chylomicron concentration correction for multiple testing (Q < 0.05). The subsequent gene-based test, was also significant (P = 0.01).
CONCLUSION - These results require replication but strongly indicate the role of LRP1 in postprandial lipoprotein uptake and/or clearance.
Copyright © 2013 Elsevier B.V. All rights reserved.
OBJECTIVE - Low-density lipoprotein-related receptor protein 1 (LRP1) is a multi-functional endocytic receptor and signaling molecule that is expressed in adipose and the hypothalamus. Evidence for a role of LRP1 in adiposity is accumulating from animal and in vitro models, but data from human studies are limited. The study objectives were to evaluate (i) relationships between LRP1 genotype and anthropometric traits, and (ii) whether these relationships were modified by dietary fatty acids.
DESIGN AND METHODS - We conducted race/ethnic-specific meta-analyses using data from 14 studies of US and European whites and 4 of African Americans to evaluate associations of dietary fatty acids and LRP1 genotypes with body mass index (BMI), waist circumference and hip circumference, as well as interactions between dietary fatty acids and LRP1 genotypes. Seven single-nucleotide polymorphisms (SNPs) of LRP1 were evaluated in whites (N up to 42 000) and twelve SNPs in African Americans (N up to 5800).
RESULTS - After adjustment for age, sex and population substructure if relevant, for each one unit greater intake of percentage of energy from saturated fat (SFA), BMI was 0.104 kg m(-2) greater, waist was 0.305 cm larger and hip was 0.168 cm larger (all P<0.0001). Other fatty acids were not associated with outcomes. The association of SFA with outcomes varied by genotype at rs2306692 (genotyped in four studies of whites), where the magnitude of the association of SFA intake with each outcome was greater per additional copy of the T allele: 0.107 kg m(-2) greater for BMI (interaction P=0.0001), 0.267 cm for waist (interaction P=0.001) and 0.21 cm for hip (interaction P=0.001). No other significant interactions were observed.
CONCLUSION - Dietary SFA and LRP1 genotype may interactively influence anthropometric traits. Further exploration of this, and other diet x genotype interactions, may improve understanding of interindividual variability in the relationships of dietary factors with anthropometric traits.
In vivo studies have shown that selenium is supplied to testis and brain by apoER2-mediated endocytosis of Sepp1. Although cultured cell lines have been shown to utilize selenium from Sepp1 added to the medium, the mechanism of uptake and utilization has not been characterized. Rat L8 myoblast cells were studied. They took up mouse Sepp1 from the medium and used its selenium to increase their glutathione peroxidase (Gpx) activity. L8 cells did not utilize selenium from Gpx3, the other plasma selenoprotein. Neither did they utilize it from Sepp1(Δ240-361), the isoform of Sepp1 that lacks the selenium-rich C-terminal domain. To identify Sepp1 receptors, a solubilized membrane fraction was passed over a Sepp1 column. The receptors apoER2 and Lrp1 were identified in the eluate by mass spectrometry. siRNA experiments showed that knockdown of apoER2, but not of Lrp1, inhibited (75)Se uptake from (75)Se-labeled Sepp1. The addition of protamine to the medium or treatment of the cells with chlorate also inhibited (75)Se uptake. Blockage of lysosome acidification did not inhibit uptake of Sepp1 but did prevent its digestion and thereby utilization of its selenium. These results indicate that L8 cells take up Sepp1 by an apoER2-mediated mechanism requiring binding to heparin sulfate proteoglycans. The presence of at least part of the selenium-rich C-terminal domain of Sepp1 is required for uptake. RT-PCR showed that mouse tissues express apoER2 in varying amounts. It is postulated that apoER2-mediated uptake of long isoform Sepp1 is responsible for selenium distribution to tissues throughout the body.
BACKGROUND/AIMS - The LDL receptor-related protein-1 gene (LRP-1) has been associated with obesity in animal models, but no such association has yet been reported in humans. As data suggest this increase in fat mass may be mediated through a mechanism involving the clearance of plasma triglyceride-rich lipoproteins (TGRL), where the LRP interacts with apolipoprotein E (ApoE) on chylomicron remnants, we aimed to examine (1) whether there was an association between 3 single nucleotide polymorphisms (SNPs) on LRP-1 with body mass index (BMI) and (2) whether any association between LRP-1 SNPs and BMI could be modified by polymorphisms on the ApoE gene when comparing the wild type ε3/ε3 genotype against mutant ApoE allele (ε2/ε4) carriers.
METHODS/RESULTS - We used data from 1,036 men and women (mean age ± SD = 49 ± 16 y) participating in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) Study. Mixed linear models, which controlled for age, sex, alcohol intake and smoking, as well as family pedigree and center of data collection were calculated. Models that used LRP-1 genotype as a predictor of BMI revealed that individuals who were homozygous for the minor allele at the LRP-1 I10701 locus had BMIs, on average, 1.03 kg/m(2) higher than major allele carriers (P = 0.03). In subsequent mixed linear models that included main effects of LRP-1 I10701 SNP and ApoE alleles, and an interaction term the two genotypes, there was no interaction detected between the LRP-1 I70701 genotype with either the ApoE ε2 or ε4 allele carriers (P>0.05).
CONCLUSIONS - This has implications for starting to understand pathways from genotype to human BMI, which may operate through TGRL uptake at the LRP-1 receptor. This may pave the way for future research into individualized dietary interventions.
Decorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β (TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a wide array of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of the TGF-β-dependent signaling pathway: LDL receptor-related protein (LRP-1) and decorin. Here we have determined the regions in decorin that are responsible for interaction with LRP-1 and are involved in TGF-β-dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptides derived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling. Furthermore, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury.