Low Density Lipoprotein Binding Research Articles

Simultaneous Rosiglitazone Release and Low-Density Lipoprotein Removal by Chondroitin Sodium Sulfate/Cyclodextrin/Poly(acrylic acid) Composite Adsorbents for Atherosclerosis Therapy.

Atherosclerosis (AS) is characterized by the accumulation of substantial low-density lipoprotein (LDL) and inflammatory response. Hemoperfusion is commonly employed for the selective removal of LDL from the body. However, conventional hemoperfusion merely focuses on LDL removal and does not address the symptom of plaque associated with AS. Based on the LDL binding properties of acrylated chondroitin sodium sulfate (CSA), acrylated beta-cyclodextrin (CD) and acrylic acid (AA), along with the anti-inflammatory property of rosiglitazone (R), the fabricated AA-CSA-CD-R microspheres could simultaneously release R and facilitate LDL removal for hemoperfusion. The AA and CSA offer electrostatic adsorption sites for LDL, while the CD provides hydrophobic adsorption sites for LDL and weak binding sites for R. According to the Sips model, the maximum static LDL adsorption capacity of AA-CSA-CD-R is determined to be 614.73 mg/g. In dynamic simulated perfusion experiments, AA-CSA-CD-R exhibits an initial cycle LDL adsorption capacity of 150.97 mg/g. The study suggests that the weakened inflammatory response favors plaque stabilization. The anti-inflammatory property of the microspheres is verified through an inflammation model, wherein the microsphere extracts are cocultured with mouse macrophages. Both qualitative analysis of iNOS\TNF-α and quantitative analysis of IL-6\TNF-α collectively demonstrate the remarkable anti-inflammatory effect of the microspheres. Therefore, the current study presents a novel blood purification treatment of eliminating pathogenic factors and introducing therapeutic factors to stabilize AS plaque.

A novel assay to measure low-density lipoproteins binding to proteoglycans.

The binding of low-density lipoprotein (LDL) to proteoglycans (PGs) in the extracellular matrix (ECM) of the arterial intima is a key initial step in the development of atherosclerosis. Although many techniques have been developed to assess this binding, most of the methods are labor-intensive and technically challenging to standardize across research laboratories. Thus, sensitive, and reproducible assay to detect LDL binding to PGs is needed to screen clinical populations for atherosclerosis risk. The aim of this study was to develop a quantitative, and reproducible assay to evaluate the affinity of LDL towards PGs and to replicate previously published results on LDL-PG binding. Immunofluorescence microscopy was performed to visualize the binding of LDL to PGs using mouse vascular smooth muscle (MOVAS) cells. An in-cell ELISA (ICE) was also developed and optimized to quantitatively measure LDL-PG binding using fixed MOVAS cells cultured in a 96-well format. We used the ICE assay to show that, despite equal APOB concentrations, LDL isolated from adults with cardiovascular disease bound to PG to a greater extent than LDL isolated from adults without cardiovascular disease (p<0.05). We have developed an LDL-PG binding assay that is capable of detecting differences in PG binding affinities despite equal APOB concentrations. Future work will focus on candidate apolipoproteins that enhance or diminish this interaction.

An Optimized Human Erythroblast Differentiation System Reveals Cholesterol-Dependency of Robust Production of Cultured Red Blood Cells Ex Vivo

The generation of cultured red blood cells (cRBCs) ex vivo represents a potentially unlimited source for RBC transfusion and other cell therapies. However, efficient differentiation and terminal maturation (symbolized by enucleation) into cRBCs ex vivo highly depend on replenishing human plasma. In addition to cost, this requirement results in variable potency across donors or batches and complicates consistent cRBC production required for clinical translations. Thus, exploration of key factor(s) in plasma and development of a compositionally defined medium would be in need. To investigate the role of human plasma in erythroblast terminal maturation, we first optimized a widely-used differentiation system that we and other have used in the past decade. We found that culture of erythroblasts in a newly developed medium called human plasma like medium (HPLM) greatly improves the yield and consistence of erythrocyte maturation. However, human serum (1-2%) was indispensable for efficient terminal maturation in the absence of plasma. The serum-derived factor or factors (likely a protein or complex) is essential for erythroblast growth assessed at day 3 after induction of terminal differentiation. We used size exclusion chromatography (SEC) and other protein biochemistry approaches to fractionate human serum and assessed the biological effect of each fraction. We found that the activity is fractions containing proteins or complexes of &amp;gt;100K Da. Proteomics revealed that the fractions with biological activities are enriched for low density lipoproteins (LDL)-containing proteins such as apolipoprotein B-100. In addition, we performed RNA-sequencing (RNA-seq) analysis on cultured erythroblasts with or without serum. Transcriptomic data revealed that the genes associated with de novo biosynthesis of steroids and cholesterol are upregulated in the absence of serum, although production of enzymes encoded by these genes alone is insufficient to provide cholesterol required for erythroblast differentiation and maturation. Hence, we hypothesize that cholesterol-carrying LDL that are naturally present in blood is sufficient to replace human serum for erythroblast survival and terminal differentiation ex vivo. We next examined directly whether LDL purified from human blood is sufficient for erythroid commitment and enucleation. Through LDL binding and uptake assays, we confirmed that LDL binds to erythroblasts that expressed the LDL receptor and is transported intracellularly. Furthermore, LDL purified from human plasma can essentially replace 2% unfractionated serum for efficient generation of erythrocytes in culture. In addition to using human erythroblasts derived from peripheral blood mononuclear cells (PBMCs), we also extended this optimized protocol to human erythroblasts from various sources, including cord blood-derived mononuclear cells, CD34+ HSPCs, and extensively expanded erythroblasts after BMI1 gene transduction. After induction of terminal differentiation for 8 days, erythroblasts from all these sources effectively enucleated (&amp;gt;50%) and stably produced reticulocytes with no significant difference compared to the serum-containing system. We are also in progress to substitute LDL by chemical synthetic cholesterol. In sum, we developed a compositionally defined culture medium (LDL-containing Optimized Maturation Medium, LOAM) enabling robust generation of cRBCs from multiple sources of erythroblasts with high enucleation efficiency and higher reticulocyte yield compared to culture in traditional media supplemented with human plasma or serum. Using this advanced human erythroblast differentiation model, we further revealed that cholesterol carried by LDL is critical to erythroblast terminal maturation.

Carbon-based light addressable potential sensor based on nitrogen-doped graphene quantum dots for detection of low-density lipoprotein

Cardiovascular diseases have become the number one killer of human health. Early diagnosis and treatment of cardiovascular diseases can effectively improve the treatment effect and reduce the disease death rate. As an important marker of cardiovascular disease, Low-Density Lipoprotein (LDL) can be used for the early detection of cardiovascular disease. In this study, a novel carbon-based Light Addressable Potentiometric Sensor (C-LAPS) for determination LDL has been constructed based on o-phenylenediamine modified nitrogen-doped graphene quantum dots/reduce graphene oxide (OPD@NGQDs/rGO) photosensitive layer. The OPD@NGQDs/rGO photosensitive layer has excellent photoelectric conversion efficiency, large specific surface area and good biocompatibility to enhance the response signal. For the determination LDL by the C-LAPS system, LDL binding to LDL aptamer causes a change in the surface potential of the photosensitive layer, producing a response signal that can be detected by the I-V curve. Under optimal conditions, the C-LAPS had a good linear relationship with LDL concentration in the range of 0.01 μg/mL- 0.15 μg/mL. The sensitivity was 1819 mV/µg·mL−1 and the limit of detection (LOD) was 6.43 ng/mL (S/N = 3). In addition, the C-LAPS showed good specificity, reproducibility, stability and satisfactory relative error for detecting LDL in human serum samples. Therefore, the C-LAPS has good performance and demonstrates potential clinical value.

Interactions of Nanoscale Self-Assembled Peptide-Based Assemblies with Glioblastoma Cell Models and Spheroids.

Peptide nanoassemblies have garnered remarkable importance in the development of novel nanoscale biomaterials for drug delivery into tumor cells. Taking advantage of receptor mediated recognition of two known peptides, angiopep-2 (TFFYGGSRGKRNNFKTEEY) and A-COOP-K (ACGLSGLC10 VAK) that bind to the over-expressed receptors low density lipoprotein (LRP-1) and fatty acid binding protein (FABP3) respectively, we have developed new peptide conjugates by combining the anti-inflammatory, antitumor compound azelaic acid with angiopep-2, which efficiently self-assembled into nanofibers. Those nanofibers were then functionalized with the A-COOP-K sequence and formed supramolecular hierarchical structures that were found to entrap the chemotherapeutic drug doxorubicin efficaciously. Furthermore, the nanoassemblies were found to release the drug in a dose-dependent manner and showed a stepwise increase over a period of 2 weeks under acidic conditions. Two cell lines (U-87-MG and U-138-MG) were utilized as models for glioblastoma cells grown in the presence of serum and under serum-free conditions to mimic the growth conditions of natural tumors. The drug entrapped assemblies were found to inhibit the cell proliferation of both U-87 and U-138MG glioblastoma cells. Three dimensional spheroids of different sizes were grown to mimic the tumors and evaluate the efficacy of drug release and internalization. Our results indicated that the nanoassemblies were found to have higher internalization of DOX and were well-spread throughout the spheroids grown, particularly under serum-free conditions. The nanoassemblies also displayed blood-brain barrier penetration when tested with a multicellular in vitro model. Such self-assembled nanostructures with targeting ability may provide a suitable platform for the development of new peptide-based biomaterials that can provide more insights about the mechanistic approach for drug delivery for not only 2D cell cultures but also 3D tumoroids that mimic the tumor microenvironments.

LDLR missense variants disturb structural conformation and LDLR activity in T-lymphocytes of Familial hypercholesterolemia patients

Familial hypercholesterolemia (FH) is caused by deleterious mutations in the LDLR that increase markedly low-density lipoprotein (LDL) cholesterol and cause premature atherosclerotic cardiovascular disease. Functional effects of pathogenic LDLR variants identified in Brazilian FH patients were assessed using in vitro and in silico studies. Variants in LDLR and other FH-related genes were detected by exon-target gene sequencing. T-lymphocytes were isolated from 26 FH patients, and 3 healthy controls and LDLR expression and activity were assessed by flow cytometry and confocal microscopy. The impact of LDLR missense variants on protein structure was assessed by molecular modeling analysis. Ten pathogenic or likely pathogenic LDLR variants (six missense, two stop-gain, one frameshift, and one in splicing region) and six non-pathogenic variants were identified. Carriers of pathogenic and non-pathogenic variants had lower LDL binding and uptake in activated T-lymphocytes compared to controls (p < 0.05), but these variants did not influence LDLR expression on cell surface. Reduced LDL binding and uptake was also observed in carriers of LDLR null and defective variants. Modeling analysis showed that p.(Ala431Thr), p.(Gly549Asp) and p.(Gly592Glu) disturb intramolecular interactions of LDLR, and p.(Gly373Asp) and p.(Ile488Thr) reduce the stability of the LDLR protein. Docking and molecular interactions analyses showed that p.(Cys184Tyr) and p.(Gly373Asp) alter interaction of LDLR with Apolipoprotein B (ApoB). In conclusion, LDLR null and defective variants reduce LDL binding capacity and uptake in activated T-lymphocytes of FH patients and LDLR missense variants affect LDLR conformational stability and dissociation of the LDLR-ApoB complex, having a potential role in FH pathogenesis.

Brain Endothelial Cells in Contrary to the Aortic Do Not Transport but Degrade Low-Density Lipoproteins via Both LDLR and ALK1.

The transport of low-density lipoprotein (LDL) through the endothelium is a key step in the development of atherosclerosis, but it is notorious that phenotypic differences exist between endothelial cells originating from different vascular beds. Endothelial cells forming the blood–brain barrier restrict paracellular and transcellular passage of plasma proteins. Here, we systematically compared brain versus aortic endothelial cells towards their interaction with LDL and the role of proteins known to regulate the uptake of LDL by endothelial cells. Both brain endothelial cells and aortic endothelial cells bind and internalize LDL. However, whereas aortic endothelial cells degrade very small amounts of LDL and transcytose the majority, brain endothelial cells degrade but do not transport LDL. Using RNA interference (siRNA), we found that the LDLR–clathrin pathway leads to LDL degradation in either endothelial cell type. Both loss- and gain-of-function experiments showed that ALK1, which promotes transcellular LDL transport in aortic endothelial cells, also limits LDL degradation in brain endothelial cells. SR-BI and caveolin-1, which promote LDL uptake and transport into aortic endothelial cells, limit neither binding nor association of LDL to brain endothelial cells. Together, these results indicate distinct LDL trafficking by brain microvascular endothelial cells and aortic endothelial cells.

PH-Dependent Protonation of Histidine Residues Is Critical for Electrostatic Binding of Low-Density Lipoproteins to Human Coronary Arteries.

The initiating step in atherogenesis is the electrostatic binding of LDL (low-density lipoprotein) to proteoglycan glycosaminoglycans in the arterial intima. However, although proteoglycans are widespread throughout the intima of most coronary artery segments, LDL is not evenly distributed, indicating that LDL retention is not merely dependent on the presence of proteoglycans. We aim to identify factors that promote the interaction between LDL and the vessel wall of human coronary arteries. We developed an ex vivo model to investigate binding of labeled human LDL to human coronary artery sections without the interference of cellular processes. By staining consecutive sections of human coronary arteries, we found strong staining of sulfated glycosaminoglycans throughout the arterial intima, whereas endogenous LDL deposits were focally distributed. Ex vivo binding of LDL was uniform at all intimal areas with sulfated glycosaminoglycans. However, lowering the pH from 7.4 to 6.5 triggered a 35-fold increase in LDL binding. The pH-dependent binding was abolished by pretreating LDL with diethyl-pyrocarbonate, which blocks the protonation of histidine residues, or cyclohexanedione, which inhibits the positive charge of site B on LDL. Thus, both histidine protonation and site B are required for strong electrostatic LDL binding to the intima. This study identifies histidine protonation as an important component for electrostatic LDL binding to human coronary arteries. Our findings show that the local pH will have a profound impact on LDL's affinity for sulfated glycosaminoglycans, which may influence the retention and accumulation pattern of LDL in the arterial vasculature.

Ultrasound-assisted pH-shifting remodels egg-yolk low-density lipoprotein to enable construction of a stable aqueous solution of vitamin D3

Egg-yolk low-density lipoprotein (LDL) has a natural liposome structure. Using ultrasound-assisted pH-shifting (pH 12), a naturally safe and stable aqueous solution of vitamin D3 (VD3) was constructed employing LDL as the carrier. Images from electron microscopy showed that pH-shifting remodeled LDL molecules, resulting in a dramatic reduction in particle size (∼50%) and an increase in specific surface area, which reduced the turbidity (27.7%) and provided new interfaces for VD3 loading. Fluorescence analyses showed that the binding of VD3 to LDL under pH-shifting was strong, involved quenching, and the binding constant was 6.19 × 104 M−1. Thermogravimetric analysis and Fourier transform-infrared spectroscopy showed that pH-shifting hydrolyzed the esters in LDL to fatty acid salts, and the maximum weight loss of LDL occurred from 381.9 °C to 457.0 °C. Ultrasonic treatment enhanced the binding of LDL and VD3 (binding constant increased to 2.56 × 107 M−1), reduced the particle size, and increased the ζ-potential of the complex between LDL and VD3, thereby resulting in the improvement of solution stability and storage stability of VD3. Ultrasound-assisted pH-shifting could remodel LDL to construct a stable aqueous solution of VD3, which showed the potential of LDL as a carrier for lipid-soluble components.

Role of Lipid Accumulation and Inflammation in Atherosclerosis: Focus on Molecular and Cellular Mechanisms.

Atherosclerosis is a chronic lipid-driven and maladaptive inflammatory disease of arterial intima. It is characterized by the dysfunction of lipid homeostasis and signaling pathways that control the inflammation. This article reviews the role of inflammation and lipid accumulation, especially low-density lipoprotein (LDL), in the pathogenesis of atherosclerosis, with more emphasis on cellular mechanisms. Furthermore, this review will briefly highlight the role of medicinal plants, long non-coding RNA (lncRNA), and microRNAs in the pathophysiology, treatment, and prevention of atherosclerosis. Lipid homeostasis at various levels, including receptor-mediated uptake, synthesis, storage, metabolism, efflux, and its impairments are important for the development of atherosclerosis. The major source of cholesterol and lipid accumulation in the arterial wall is proatherogenic modified low-density lipoprotein (mLDL). Modified lipoproteins, such as oxidized low-density lipoprotein (ox-LDL) and LDL binding with proteoglycans of the extracellular matrix in the intima of blood vessels, cause aggregation of lipoprotein particles, endothelial damage, leukocyte recruitment, foam cell formation, and inflammation. Inflammation is the key contributor to atherosclerosis and participates in all phases of atherosclerosis. Also, several studies have shown that microRNAs and lncRNAs have appeared as key regulators of several physiological and pathophysiological processes in atherosclerosis, including regulation of HDL biogenesis, cholesterol efflux, lipid metabolism, regulating of smooth muscle proliferation, and controlling of inflammation. Thus, both lipid homeostasis and the inflammatory immune response are closely linked, and their cellular and molecular pathways interact with each other.

Study of serum obestatin level in type 2 diabetic patients with diabetic kidney disease

Objective The aim was to evaluate serum obestatin level in type 2 diabetic patients with diabetic kidney disease as a diagnostic and a prognostic marker. Background Obestatin has been reported to have important effects on endothelial cells as decreasing vascular cell adhesion molecule-1 expression and increasing oxidized low-density lipoprotein binding to the macrophage. Participant and methods A cross-sectional study was carried out on 100 patients sin the Department of Internal Medicine, Menoufia University Hospitals. A total of 100 patients was divided into different groups. Group I: 25 type 2 diabetic patients with normoalbuminuric; group II: 25 type 2 diabetic patients with microalbuminuria; group III: 25 type 2 diabetic patients with macroalbuminuria; and group IV: 25 healthy individuals as a control group. All participants were subjected to history taking, complete physical examination, anthropometric measurement, waist circumferences, fasting blood glucose, 2h-postprandial blood glucose, glycated hemoglobin, kidney function tests, estimated glomerular filtration rate, urine analysis, abdominal ultrasound, liver profile, lipid profile, complete blood count, and serum obestatin level by ELIZA kit. Results Obestatin was 2.9 ± 0.4 ng/dl in group I, 3.7 ± 0.7 g/dl in group II, 5.5 ± 0.9 g/dl in group III, and 2.4 ± 0.6 g/dl in group VI. These results were significantly higher in group III than in groups I, II, and VI (P Conclusion Higher serum levels of obestatin were associated with macroalbuminuria, suggesting that obestatin may have a role in the underlying pathogenic mechanisms that lead to diabetic kidney disease.

Dyslipidemia in Chinese Pancreatic Cancer Patients: A Two-Center Retrospective Study.

Background: Pancreatic cancer (PC) is one of the most aggressive and lethal malignancies in the world. High cholesterol intake may have a certain association with an elevated risk of PC, though dyslipidemia in PC patients has rarely been reported. In this study, we compared serum lipids levels between PC and non-PC tumor patients and assessed their prognostic value in PC.Methods: 271 patients treated at Wuhan Union Hospital from January 2012 to December 2016 and 204 individuals at Shanghai General Hospital from January 2018 to December 2019 were recruited. Their demographic parameters, laboratory data, pathological information, and clinical outcomes were extracted and analyzed. The mRNA expressions of related lipoprotein, low density lipoprotein receptor (LDLR) and high density lipoprotein binding protein (HDLBP), in PC tissues and paired noncancerous tissues and follow-up information were assessed based on the GEO database (GSE15471 and GSE62165) and TCGA database.Results: A total of 172 non-PC tumor patients and 260 PC patients were finally eligible for our analysis. PC patients exhibited higher levels of serum triglyceride, cholesterol, and low-density lipoprotein (LDL) and a lower serum high-density lipoprotein (HDL) level on admission versus the non-PC tumor group. In PC patients, LDLR mRNA expression was upregulated, and HDLBP mRNA expression was downregulated in cancerous tissues compared to these levels in paired noncancerous tissues. The survival analysis revealed that dyslipidemia had a non-significant association with a poor prognosis, but PC patients with a high LDLR level were at risk of poor survival.Conclusion: Dyslipidemia is detected in PC patients but has a non-significant relation to PC prognosis. However, LDLR may be a potential predictive marker for PC prognosis.

Macrophages bind LDL using heparan sulfate and the perlecan protein core

The retention of low-density lipoprotein (LDL) is a key process in the pathogenesis of atherosclerosis and largely mediated via smooth-muscle cell-derived extracellular proteoglycans including the glycosaminoglycan chains. Macrophages can also internalize lipids via complexes with proteoglycans. However, the role of polarized macrophage-derived proteoglycans in binding LDL is unknown and important to advance our understanding of the pathogenesis of atherosclerosis. We therefore examined the identity of proteoglycans, including the pendent glycosaminoglycans, produced by polarized macrophages to gain insight into the molecular basis for LDL binding. Using the quartz crystal microbalance with dissipation monitoring technique, we established that classically activated macrophage (M1)- and alternatively activated macrophage (M2)-derived proteoglycans bind LDL via both the protein core and heparan sulfate (HS) in vitro. Among the proteoglycans secreted by macrophages, we found perlecan was the major protein core that bound LDL. In addition, we identified perlecan in the necrotic core as well as the fibrous cap of advanced human atherosclerotic lesions in the same regions as HS and colocalized with M2 macrophages, suggesting a functional role in lipid retention in vivo. These findings suggest that macrophages may contribute to LDL retention in the plaque by the production of proteoglycans; however, their contribution likely depends on both their phenotype within the plaque and the presence of enzymes, such as heparanase, that alter the secreted protein structure.

The α-defensins stimulate proteoglycan-dependent catabolism of low-density lipoprotein by vascular cells: a new class of inflammatory apolipoprotein and a possible contributor to atherogenesis

AbstractInflammation may contribute to the pathogenesis of atherosclerosis. On the basis of previous reports that human atherosclerotic lesions contain α-defensins, a class of cationic proteins released by activated neutrophils, the study was designed to ask whether defensins modulate the binding and catabolism of low-density lipoprotein (LDL) by human vascular cells. The results of the study demonstrated that defensin stimulated the binding of 125I-LDL to cultured human umbilical vein endothelial cells, smooth muscle cells, and fibroblasts approximately 5-fold in a dose-dependent and saturable manner. Defensin and LDL formed stable complexes in solution and on cell surfaces. Stimulation of LDL binding by defensin was not inhibited by antibodies against the LDL-receptor (LDL-R), or by recombinant receptor-associated protein, which blocks binding of ligands to the α2-macroglobulin receptor/LDL-R–related protein and other LDL-R family members. Furthermore, defensin stimulated the binding, endocytosis, and degradation of LDL by fibroblasts lacking LDL-R. Stimulation of LDL degradation by defensin was inhibited approximately 75% by low concentrations of heparin (0.2 units/mL) and was similarly reduced in CHO cells lacking heparan-sulfate–containing proteoglycans. The effect of defensin was substantially increased in cells overexpressing the core protein of the syndecan-1 heparan sulfate proteoglycan. The α-defensins released from activated neutrophils may provide a link between inflammation and atherosclerosis by changing the pattern of LDL catabolism from LDL-R to the less efficient LDL-R–independent, proteoglycan-dependent pathway.

Binding of Macrophage Receptor MARCO, LDL, and LDLR to Disease-Associated Crystalline Structures.

Endogenous and exogenous crystalline structures are involved in various pathologies and diseases in humans by inducing sterile inflammation, mechanical stress, or obstruction of excretory organs. The best studied of these diseases is gout, in which crystallization of uric acid in the form of monosodium urate (MSU) mainly in synovial fluid of the joints leads to sterile inflammation. Though some of these diseases have been described for centuries, little is known about if and how the immune system recognizes the associated crystals. Thus, in this study we aimed at identifying possible recognition molecules of MSU using liquid chromatography-mass spectrometry (LC-MS) analysis of MSU-binding serum proteins. Among the strongest binding proteins, we unexpectedly found two transmembrane receptors, namely macrophage receptor with collagenous structure (MARCO) and low-density lipoprotein (LDL) receptor (LDLR). We show that recombinant versions of both human and mouse MARCO directly bind to unopsonized MSU and several other disease-associated crystals. Recombinant LDLR binds many types of crystals mainly when opsonized with serum proteins. We show that this interaction is predominantly mediated by LDL, which we found to bind to all crystalline structures tested except for cholesterol crystals. However, murine macrophages lacking LDLR expression do neither show altered phagocytosis nor interleukin-1β (IL-1β) production in response to opsonized crystals. Binding of LDL to MSU has previously been shown to inhibit the production of reactive oxygen species (ROS) by human neutrophils. We extend these findings and show that LDL inhibits neutrophil ROS production in response to most crystals tested, even cholesterol crystals. The inhibition of neutrophil ROS production only partly correlated with the inhibition of IL-1β production by peripheral blood mononuclear cells (PBMCs): LDL inhibited IL-1β production in response to large MSU crystals, but not small MSU or silica crystals. This may suggest distinct upstream signals for IL-1β production depending on the size or the shape of the crystals. Together, we identify MARCO and LDLR as potential crystal recognition receptors, and show that LDL binding to diverse disease-associated crystalline structures has variable effects on crystal-induced innate immune cell activation.

Brain metastases-derived extracellular vesicles induce binding and aggregation of low-density lipoprotein

BackgroundCancer cell-derived extracellular vesicles (EVs) have previously been shown to contribute to pre-metastatic niche formation. Specifically, aggressive tumors secrete pro-metastatic EVs that travel in the circulation to distant organs to modulate the microenvironment for future metastatic spread. Previous studies have focused on the interface between pro-metastatic EVs and epithelial/endothelial cells in the pre-metastatic niche. However, EV interactions with circulating components such as low-density lipoprotein (LDL) have been overlooked.ResultsThis study demonstrates that EVs derived from brain metastases cells (Br-EVs) and corresponding regular cancer cells (Reg-EVs) display different interactions with LDL. Specifically, Br-EVs trigger LDL aggregation, and the presence of LDL accelerates Br-EV uptake by monocytes, which are key components in the brain metastatic niche.ConclusionsCollectively, these data are the first to demonstrate that pro-metastatic EVs display distinct interactions with LDL, which impacts monocyte internalization of EVs.

Reductions of copper ion-mediated low-density lipoprotein (LDL) oxidations of trypsin inhibitors, the sweet potato root major proteins, and LDL binding capacities

BackgroundThe root major proteins of sweet potato trypsin inhibitors (SPTIs) or named sporamin, estimated for 60 to 80% water-soluble proteins, exhibited many biological activities. The human low-density lipoprotein (LDL) showed to form in vivo complex with endogenous oxidized alpha-1-antitrypsin. Little is known concerning the interactions between SPTIs and LDL in vitro.ResultsThe thiobarbituric-acid-reactive-substance (TBARS) assays were used to monitor 0.1 mM Cu2+-mediated low-density lipoprotein (LDL) oxidations during 24-h reactions with or without SPTIs additions. The protein stains in native PAGE gels were used to identify the bindings between native or reduced forms of SPTIs or soybean TIs and LDL, or oxidized LDL (oxLDL). It was found that the SPTIs additions showed to reduce LDL oxidations in the first 6-h and then gradually decreased the capacities of anti-LDL oxidations. The protein stains in native PAGE gels showed more intense LDL bands in the presence of SPTIs, and 0.5-h and 1-h reached the highest one. The SPTIs also bound to the oxLDL, and low pH condition (pH 2.0) might break the interactions revealed by HPLC. The LDL or oxLDL adsorbed onto self-prepared SPTIs-affinity column and some components were eluted by 0.2 M KCl (pH 2.0). The native or reduced SPTIs or soybean TIs showed different binding capacities toward LDL and oxLDL in vitro.ConclusionThe SPTIs might be useful in developing functional foods as antioxidant and nutrient supplements, and the physiological roles of SPTIs-LDL and SPTIs-oxLDL complex in vivo will investigate further using animal models.

Plant Stanol Esters Reduce LDL (Low-Density Lipoprotein) Aggregation by Altering LDL Surface Lipids: The BLOOD FLOW Randomized Intervention Study.

Plant stanol ester supplementation (2-3 g plant stanols/d) reduces plasma LDL (low-density lipoprotein) cholesterol concentration by 9% to 12% and is, therefore, recommended as part of prevention and treatment of atherosclerotic cardiovascular disease. In addition to plasma LDL-cholesterol concentration, also qualitative properties of LDL particles can influence atherogenesis. However, the effect of plant stanol ester consumption on the proatherogenic properties of LDL has not been studied. Approach and Results: Study subjects (n=90) were randomized to consume either a plant stanol ester-enriched spread (3.0 g plant stanols/d) or the same spread without added plant stanol esters for 6 months. Blood samples were taken at baseline and after the intervention. The aggregation susceptibility of LDL particles was analyzed by inducing aggregation of isolated LDL and following aggregate formation. LDL lipidome was determined by mass spectrometry. Binding of serum lipoproteins to proteoglycans was measured using a microtiter well-based assay. LDL aggregation susceptibility was decreased in the plant stanol ester group, and the median aggregate size after incubation for 2 hours decreased from 1490 to 620 nm, P=0.001. Plant stanol ester-induced decrease in LDL aggregation was more extensive in participants having body mass index<25 kg/m2. Decreased LDL aggregation susceptibility was associated with decreased proportion of LDL-sphingomyelins and increased proportion of LDL-triacylglycerols. LDL binding to proteoglycans was decreased in the plant stanol ester group, the decrease depending on decreased serum LDL-cholesterol concentration. Consumption of plant stanol esters decreases the aggregation susceptibility of LDL particles by modifying LDL lipidome. The resulting improvement of LDL quality may be beneficial for cardiovascular health. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01315964.

LDL Receptor Pathway Regulation by miR-224 and miR-520d.

MicroRNAs (miRNA) have emerged as important post-transcriptional regulators of metabolic pathways that contribute to cellular and systemic lipoprotein homeostasis. Here, we identify two conserved miRNAs, miR-224, and miR-520d, which target gene networks regulating hepatic expression of the low-density lipoprotein (LDL) receptor (LDLR) and LDL clearance. In silico prediction of miR-224 and miR-520d target gene networks showed that they each repress multiple genes impacting the expression of the LDLR, including the chaperone molecules PCSK9 and IDOL that limit LDLR expression at the cell surface and the rate-limiting enzyme for cholesterol synthesis HMGCR, which is the target of LDL-lowering statin drugs. Using gain- and loss-of-function studies, we tested the role of miR-224 and miR-520d in the regulation of those predicted targets and their impact on LDLR expression. We show that overexpression of miR-224 or miR-520d dose-dependently reduced the activity of PCSK9, IDOL, and HMGCR 3′-untranslated region (3′-UTR)-luciferase reporter constructs and that this repression was abrogated by mutation of the putative miR-224 or miR-520d response elements in the PCSK9, IDOL, and HMGCR 3′-UTRs. Compared to a control miRNA, overexpression of miR-224 or miR-520d in hepatocytes inhibited PCSK9, IDOL, and HMGCR mRNA and protein levels and decreased PCSK9 secretion. Furthermore, miR-224 and miR-520d repression of PCSK9, IDOL, and HMGCR was associated with an increase in LDLR protein levels and cell surface expression, as well as enhanced LDL binding. Notably, the effects of miR-224 and miR-520d were additive to the effects of statins in upregulating LDLR expression. Finally, we show that overexpression of miR-224 in the livers of Ldlr+/− mice using lipid nanoparticle-mediated delivery resulted in a 15% decrease in plasma levels of LDL cholesterol, compared to a control miRNA. Together, these findings identify roles for miR-224 and miR-520d in the posttranscriptional control of LDLR expression and function.

Binding to heparin triggers deleterious structural and biochemical changes in human low-density lipoprotein, which are amplified in hyperglycemia

Low-density lipoprotein (LDL) binding to arterial proteoglycans initiates LDL retention and modification in the arterial wall, triggering atherosclerosis. The details of this binding, its effectors, and its ramifications are incompletely understood. We combined heparin affinity chromatography with biochemical, spectroscopic and electron microscopic techniques to show that brief binding to heparin initiates irreversible pro-atherogenic remodeling of human LDL. This involved decreased structural stability of LDL and increased susceptibility to hydrolysis, oxidation and fusion. Furthermore, phospholipid hydrolysis, mild oxidation and/or glycation of LDL in vitro increase the proteolytic susceptibility of apoB and its heparin binding affinity, perhaps by unmasking additional heparin-binding sites. For LDL from hyperglycemic type-2 diabetic patients, heparin binding was particularly destabilizing and caused apoB fragmentation and LDL fusion. However, for similar patients whose glycemic control was restored upon therapy, LDL-heparin binding affinity was rectified and LDL structural stability was partially restored. These results complement previous studies of LDL binding to arterial proteoglycans and suggest that such interactions may produce a particularly pro-atherogenic subclass of electronegative LDL. In summary, binding to heparin alters apoB conformation, perhaps by partially peeling it off the lipid, and triggers pro-atherogenic LDL modifications including hydrolysis, oxidation, and destabilization. Furthermore, phospholipid lipolysis, mild oxidation and glycation of LDL in vitro strengthen its binding to heparin, which helps explain stronger binding observed in hyperglycemic LDL. Combined effects of hyperglycemia and heparin binding are especially deleterious but are largely rectified upon diabetes therapy. These findings help establish a mechanistic link between diabetes and atherosclerosis.