Apo(a) and ApoB Interact Noncovalently Within Hepatocytes: Implications for Regulation of Lp(a) Levels by Modulation of ApoB Secretion.

BACKGROUND: Elevated plasma Lp(a) (lipoprotein(a)) levels are associated with increased risk for atherosclerotic cardiovascular disease and aortic valve stenosis. However, the cell biology of Lp(a) biosynthesis remains poorly understood, with the locations of the noncovalent and covalent steps of Lp(a) assembly unclear and the nature of the apoB-containing particle destined for Lp(a) unknown. We, therefore, asked if apo(a) and apoB interact noncovalently within hepatocytes and if this impacts Lp(a) biosynthesis. METHODS: Using human hepatocellular carcinoma cells expressing 17K (17 kringle) apo(a), or a 17KΔLBS7,8 variant with a reduced ability to bind noncovalently to apoB, we performed coimmunoprecipitation, coimmunofluorescence, and proximity ligation assays to document intracellular apo(a):apoB interactions. We used a pulse-chase metabolic labeling approach to measure apo(a) and apoB secretion rates. RESULTS: Noncovalent complexes containing apo(a)/apoB are present in lysates from cells expressing 17K but not 17KΔLBS7,8, whereas covalent apo(a)/apoB complexes are absent from lysates. 17K and apoB colocalized intracellularly, overlapping with staining for markers of endoplasmic reticulum trans-Golgi, and early endosomes, and less so with lysosomes. The 17KΔLBS7,8 had lower colocalization with apoB. Proximity ligation assays directly documented intracellular 17K/apoB interactions, which were dramatically reduced for 17KΔLBS7,8. Treatment of cells with PCSK9 (proprotein convertase subtilisin/kexin type 9) enhanced, and lomitapide reduced, apo(a) secretion in a manner dependent on the noncovalent interaction between apo(a) and apoB. Apo(a) secretion was also reduced by siRNA-mediated knockdown of APOB. CONCLUSIONS: Our findings explain the coupling of apo(a) and Lp(a)-apoB production observed in human metabolic studies using stable isotopes as well as the ability of agents that inhibit apoB biosynthesis to lower Lp(a) levels.

siRNA potency enhancement via chemical modifications of nucleotide bases at the 5'-end of the siRNA guide strand.

Small interfering RNAs (siRNAs) can be utilized not only as functional biological research tools but also as therapeutic agents. For the clinical use of siRNA as drugs, various chemical modifications have been used to improve the activity of siRNA drugs, and further chemical modifications are expected to improve the utility of siRNA therapeutics. As the 5' nucleobase of the guide strand affects the interaction between an siRNA and AGO2 and target cleavage activity, structural optimization of this specific position may be a useful strategy for improving siRNA activity. Here, using the in silico model of the complex between human AGO2 MID domain and nucleoside monophosphates, we screened and synthesized an original adenine-derived analog, 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), that fits better than the natural nucleotide bases into the MID domain of AGO2. Introduction of the 6-mCEPh-purine analog at the 5'-end of the siRNA guide strand significantly enhanced target knockdown activity in both cultured cell lines and in vivo animal models. Our findings can help expand strategies for rationally optimizing siRNA activity via chemical modifications of nucleotide bases.

Induction of HLA-A2 restricted CD8 T cell responses against ApoB100 peptides does not affect atherosclerosis in a humanized mouse model.

Cardiovascular diseases form the most common cause of death worldwide, with atherosclerosis as main etiology. Atherosclerosis is marked by cholesterol rich lipoprotein deposition in the artery wall, evoking a pathogenic immune response. Characteristic for the disease is the pathogenic accumulation of macrophages in the atherosclerotic lesion, which become foam cells after ingestion of large quantities of lipoproteins. We hypothesized that, by inducing a CD8 T cell response towards lipoprotein derived apolipoprotein-B100 (ApoB100), lesional macrophages, that are likely to cross-present lipoprotein constituents, can specifically be eliminated. Based on in silico models for protein processing and MHC-I binding, 6 putative CD8 T cell epitopes derived from ApoB100 were synthesized. HLA-A2 binding was confirmed for all peptides by T2 cell binding assays and recall responses after vaccination with the peptides proved that 5 of 6 peptides could induce CD8 T cell responses. Induction of ApoB100 specific CD8 T cells did not impact plaque size and cellular composition in HLA-A2 and human ApoB100 transgenic LDLr-/- mice. No recall response could be detected in cultures of cells isolated from the aortic arch, which were observed in cell cultures of splenocytes and mesenteric lymph nodes, suggesting that the atherosclerotic environment impairs CD8 T cell activation.

Low density lipoprotein mimic nanoparticles composed of amphipathic hybrid peptides and lipids for tumor-targeted delivery of paclitaxel.

BACKGROUND: Low density lipoprotein (LDL) has been regarded as a promising antitumor drug vehicle. However some problems, such as rare source, difficulty of large-scale production, and potential safety concerns, hinder its clinical application. PURPOSE: The objective of this study is to develop a biomimetic LDL nanocarrier by replacing the native apolipoprotein B-100 (apoB-100) with an artificial amphipathic peptide and demonstrate its antitumor efficacy. METHODS: The amphipathic hybrid peptide (termed as FPL) consisting of a lipid binding motif of apoB-100 (LBMapoB)-polyethylene glycol (PEG)-folic acid (FA) was synthesized and characterized by 1H NMR and circular dichroism. FPL decorated lipoprotein-mimic nanoparticles (termed as FPLM NPs) were prepared by a modified solvent emulsification method. Paclitaxel (PTX) was incorporated into NPs and its content was quantified by HPLC analysis. The morphology of NPs was observed by transmission electron microscopy (TEM), and the particle size and zeta potential of NPs were determined by dynamic light scattering (DLS). The colloidal stability of FPLM NPs was evaluated in PBS containing bovine serum albumin (BSA). In vitro release of PTX loaded FPLM NPs was evaluated using the dialysis method. Cellular uptake and cytotoxity assayswere evaluated on human cervical cancer cells (HeLa) and lung cancer cells (A549). Tumor inhibition in vivo was investigated in M109 tumor-bearing mice via tail vein injection of Taxol formulation and PTX loaded NPs. RESULTS: The composition of FPLM NPs, including cholesteryl oleate, glyceryl trioleate, cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and FPL peptides, was optimized to be 5:1:1:3:10 (w/w). FPLM NPs had a spherical shape with a mean diameter of 83 nm and a negative charge (-12 mV). FPLM NPs with optimum formulation had good colloidal stability in BSA solution.The release of PTX from FPLM NPs was slow and sustained. The uptake of FPLM NPs was higher in folate receptor (FR) overexpressing tumor cells (HeLa cells) than in FR deficient tumor cells (A549 cells). The intracellular distribution indicated that FPLM NPs had the lysosome escape capacity. The internalization mechanism of FPLM NPs was involved with clathrin- and caveolae-mediated endocytosis and FR played a positive role in the internalization of FPLM NPs. The CCK-8 assay demonstrated that FPLM NPs exhibited notably better anti-tumor effect than Taxol formulation in vitro. Moreover, PTX loaded FPLM NPs produced very marked anti-tumor efficiency in M109 tumor-bearing mice in vivo. CONCLUSION: FPLM NPs is a promising nanocarrier which can improve the therapeutic effect and reduce the side effects of antitumor drugs.

Identification of Receptor Ligands in Apo B100 Reveals Potential Functional Domains.

LDL, VLDL and other members of the low-density lipoparticles (LLPs) enter cells through a large family of receptors. The actual receptor ligand(s) in apolipoprotein B100, one of the main proteins of LLP, remain(s) unknown. The objective of this study was to identify true receptor ligand(s) in apo B100, a molecule of 4563 residues. Apo B100 contains 33 analogues of Cardin-Weintraub arginine/lysine-based receptor ligand motifs and shares key lysine motifs and sequence similarity with the LDL receptor-associated protein, MESD, and heat shock proteins. Eleven FITC-labeled synthetic peptides of 21-42 residues, with at least one ligand, were tested for binding and internalization using HeLa cells. All peptides bind but display different binding capacities and patterns. Peptides B0013, B0582, B2366, and B2932 mediate endocytosis and appear in distinct sites in the cytoplasm. B0708 and B3181 bind and remain on the cell surface as aggregates/clusters. Peptides B3119 (Site A) and B3347 (Site B), the putative ligands, showed low binding and no cell entry capacity. Apo B100 regions in this study share similarities with related proteins of known function including chaperone proteins and Apo BEC stimulating protein, and not directly related proteins, e.g., the DNA-binding domain of interferon regulatory factors, MSX2-interacting protein, and snake venom Zinc metalloproteinase-disintegrin-like proteins.

LDL dinitrosyl iron complex acts as an iron donor in mouse macrophages.

[Fe(NO)2] - modified nanoparticles of low-density protein (DNICLDL) can serve as conveyors of iron in the form of stable complexes with ApoB100 protein. As reported recently, in human hepatoma cells DNICLDL significantly increased the total iron content, while showing low toxicity. In the present work, we focused on the effects of internalization of DNIC-modified lipoproteins in macrophages, with special regards to cytotoxicity. DNICLDL was administered to a model macrophage cell line, RAW 264.7. Administration of DNICLDL considerably increased total iron content. High increase of iron was accompanied by moderate toxicity. As shown by in vitro plasmid nicking assay, chelation of iron in the form of DNIC strongly reduced the iron-related reactive oxygen species (ROS) -induced DNA damage. In addition, DNICLDL, plausibly due to its NO-donating activity, did not induce inducible nitric oxide synthase (iNOS) expression, as opposed to other forms of low-density protein (LDL).

Relationship between Amphipathic ß Structures in the ß1 Domain of Apolipoprotein B and the Properties of the Secreted Lipoprotein Particles in McA-RH7777 Cells.

Our previous studies demonstrated that the first 1000 amino acid residues (the ßα1 domain) of human apolipoprotein (apo) B-100, termed apoB:1000, are required for the initiation of lipoprotein assembly and the formation of a monodisperse stable phospholipid (PL)-rich particle. The objectives of this study were (a) to assess the effects on the properties of apoB truncates undergoing sequential inclusion of the amphipathic ß strands in the 700 N-terminal residues of the ß1 domain of apoB-100 and (b) to identify the subdomain in the ß1 domain that is required for the formation of a microsomal triglyceride transfer protein (MTP)-dependent triacylglycerol (TAG)-rich apoB-containing particle. Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following. (1) The presence of amphipathic ß strands in the 200 N-terminal residues of the ß1 domain resulted in the secretion of apoB truncates (apoB:1050 to apoB:1200) as both lipidated and lipid-poor particles. (2) Inclusion of residues 300-700 of the ß1 domain led to the secretion of apoB:1300, apoB:1400, apoB:1500, and apoB:1700 predominantly as lipidated particles. (3) Particles containing residues 1050-1500 were all rich in PL. (4) There was a marked increase in the lipid loading capacity and TAG content of apoB:1700-containing particles. (5) Only the level of secretion of apoB:1700 was markedly diminished by MTP inhibitor BMS-197636. These results suggest that apoB:1700 marks the threshold for the formation of a TAG-rich particle and support the concept that MTP participates in apoB assembly and secretion at the stage where particles undergo a transition from PL-rich to TAG-rich.

Separation and characterization of oxidized isomeric lipid-peptide adducts by ion mobility mass spectrometry.

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid-protein adducts by liquid chromatography-MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid-peptide adducts generated by incubating model peptides corresponding to the amphipathic ß1 sheet sequence of apolipoprotein B-100 with 1-palmitoyl-2-(oxo-nonanoyl)-sn-glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide-lipid adducts was separated by TWIMS, which was especially important for the identification of two mono-PONPC-peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide-lipid adduct possessing most likely different orientations of the hydrophobic sn-1 fatty acyl residue and head group of PONPC, relative to the peptide backbone.

Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicity.

Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease.

The intravenous injection of oxidized LDL- or Apolipoprotein B100--Coupled splenocytes promotes Th1 polarization in wildtype and Apolipoprotein E--Deficient mice.

BACKGROUND: Th1 responses in atherosclerosis are mainly associated with the aggravation of atherosclerotic plaques, whereas Th2 responses lead to a less pronounced disease in mouse models. The fixation of antigens on cells by means of ethylene carbodiimide (ECDI), and subsequent injection of these antigen-coupled splenocytes (Ag-SP) to induce tolerance against the attached antigens, has been successfully used to treat murine type 1 diabetes or encephalomyelitis in. We analyzed this approach in a mouse model for atherosclerosis. METHODS AND RESULTS: OTII-transgenic mice that were treated with a single dose of 5 × 10(7) OVA-coupled splenocytes (OVA-SP), had decreased splenocyte proliferation, and lower IFNγ production in vitro upon antigen recall. However, in vivo CD4 cell activation was increased. To try lipoprotein-derived, "atherosclerosis-associated" antigens, we first tested human oxidized LDL. In wild type mice, an increase of IFNγ production upon in vitro recall was detected in the oxLDL-SP group. In Apolipoprotein E - deficient (ApoE-/-) mice that received oxLDL-SP every 5 weeks for 20 weeks, we did not find any difference of atherosclerotic plaque burden, but again increased IFNγ production. To overcome xenogenous limitations, we then examined the effects of mouse Apolipoprotein B100 peptides P3 and P6. ApoB100-SP treatment again promoted a more IFNγ pronounced response upon in vitro recall. Flow cytometry analysis of cytokine secreting spleen cells revealed CD4 positive T cells to be mainly the source for IFNγ. In ApoE-/- mice that were administered ApoB100-SP during 20 weeks, the atherosclerotic plaque burden in aortic roots as well as total aorta was unchanged compared to PBS treated controls. Splenocyte proliferation upon antigen recall was not significantly altered in ApoB100-SP treated ApoE-/- mice. CONCLUSION: Although we did not observe a relevant anti-atherosclerotic benefit, the treatment with antigen-coupled splenocytes in its present form already impacts the immune responses and deserves further exploration.

Immunization with an ApoB-100 Related Peptide Vaccine Attenuates Angiotensin-II Induced Hypertension and Renal Fibrosis in Mice.

Recent studies suggest the potential involvement of CD8+ T cells in the pathogenesis of murine hypertension. We recently reported that immunization with apoB-100 related peptide, p210, modified CD8+ T cell function in angiotensin II (AngII)-infused apoE (-/-) mice. In this study, we hypothesized that p210 vaccine modulates blood pressure in AngII-infused apoE (-/-) mice. Male apoE (-/-) mice were immunized with p210 vaccine and compared to unimmunized controls. At 10 weeks of age, mice were subcutaneously implanted with an osmotic pump which released AngII for 4 weeks. At 13 weeks of age, p210 immunized mice showed significantly lower blood pressure response to AngII compared to controls. CD8+ T cells from p210 immunized mice displayed a different phenotype compared to CD8+ T cells from unimmunized controls. Serum creatinine and urine albumin to creatinine ratio were significantly decreased in p210 immunized mice suggesting that p210 vaccine had renal protective effect. At euthanasia, inflammatory genes IL-6, TNF-α, and MCP-1 in renal tissue were down-regulated by p210 vaccine. Renal fibrosis and pro-fibrotic gene expression were also significantly reduced in p210 immunized mice. To assess the role of CD8+ T cells in these beneficial effects of p210 vaccine, CD8+ T cells were depleted by CD8 depleting antibody in p210 immunized mice. p210 immunized mice with CD8+ T cell depletion developed higher blood pressure compared to mice receiving isotype control. Depletion of CD8+ T cells also increased renal fibrotic gene expression compared to controls. We conclude that immunization with p210 vaccine attenuated AngII-induced hypertension and renal fibrosis. CD8+ T cells modulated by p210 vaccine could play an important role in the anti-hypertensive, anti-fibrotic and renal-protective effect of p210 vaccine.

Comparative reactivity of the myeloperoxidase-derived oxidants HOCl and HOSCN with low-density lipoprotein (LDL): Implications for foam cell formation in atherosclerosis.

Atherosclerosis is characterised by the accumulation of lipids within macrophages in the artery wall. Low-density lipoprotein (LDL) is the source of this lipid, owing to the uptake of oxidised LDL by scavenger receptors. Myeloperoxidase (MPO) released by leukocytes during inflammation produces oxidants that are implicated in atherosclerosis. Modification of LDL by the MPO oxidant hypochlorous acid (HOCl), results in extensive lipid accumulation by macrophages. However, the reactivity of the other major MPO oxidant, hypothiocyanous acid (HOSCN) with LDL is poorly characterised, which is significant given that thiocyanate is the favoured substrate for MPO. In this study, we comprehensively compare the reactivity of HOCl and HOSCN with LDL, and show key differences in the profile of oxidative damage observed. HOSCN selectively modifies Cys residues on apolipoprotein B100, and oxidises cholesteryl esters resulting in formation of lipid hydroperoxides, 9-hydroxy-10,12-octadecadienoic acid (9-HODE) and F2-isoprostanes. The modification of LDL by HOSCN results macrophage lipid accumulation, though generally to a lesser extent than HOCl-modified LDL. This suggests that a change in the ratio of HOSCN:HOCl formation by MPO from variations in plasma thiocyanate levels, will influence the nature of LDL oxidation in vivo, and has implications for the progression of atherosclerosis.

Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100.

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding-impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.

Serum protein profiling of adults and children with Crohn disease.

OBJECTIVES: Crohn disease (CD) and ulcerative colitis (UC), known collectively as inflammatory bowel diseases (IBDs), are chronic immunoinflammatory pathologies of unknown aetiology. Despite the frequent use of biomarkers in medical practice, there is a relative lack of information regarding validated paediatric biomarkers for IBD. Furthermore, biomarkers proved to be efficacious in adults are frequently extrapolated to the paediatric clinical setting without considering that the pathogenesis of many diseases is distinctly different in children. In the present study, proteomics technology was used to monitor differences in protein expression among adult and young patients with CD, identify a panel of candidate protein biomarkers that may be used to improve prognostic-diagnostic accuracy, and advance paediatric medical care. METHODS: Male and female serum samples from 12 adults and 12 children with active CD were subjected to 2-dimensional gel electrophoresis. Following the relative quantitation of protein spots exhibiting a differential expression between the 2 groups by densitometry, the spots were further characterized by matrix-assisted laser desorption tandem time-of-flight mass spectrometer. The results were confirmed by Western blot analysis. RESULTS: Clusterin was found to be significantly overexpressed in adults with CD, whereas ceruloplasmin and apolipoprotein B-100 were found to be significantly overexpressed in children, indicating that the expression of these proteins may be implicated in the onset or progression of CD in these 2 subgroups of patients. CONCLUSIONS: Interestingly, we found a differential expression of several proteins in adults versus paediatric patients with CD. Undoubtedly, future experiments using a larger cohort of patients with CD are needed to evaluate the relevance of our preliminary findings.

Proatherogenic modification of LDL by surface-bound myeloperoxidase.

One of the factors promoting oxidative/halogenating modification of low-density lipoproteins (LDL) is myeloperoxidase (MPO). We have shown previously that MPO binds to the LDL surfaces. The LDL-MPO complex is uncoupled in the presence of peptide EQIQDDCTGDED that corresponds to a fragment of apoB-100 (445-456). In this paper we studied how this peptide, as well as inhibitors and modulators of halogenating activity of MPO such as ceruloplasmin (CP), 4-aminobenzoic acid hydrazide (ABAH) and thiocyanate (SCN(-)) affect the accumulation of cholesterol and its esters in monocytes/macrophages after incubation with LDL subjected to different kinds of MPO-dependent oxidative/halogenating modification. In the presence of H2O2 and halides MPO causes stronger proatherogenic modification of LDL than exogenous reactive halogen species (HOCl and HOBr). Both monocytes, which differentiate into macrophages, and neutrophils secrete MPO in response to the presence of damaged LDL. The peptide EQIQDDCTGDED preventing interaction between MPO and LDL reduces the uptake of modified LDL and MPO by monocytes/macrophages and thus precludes the accumulation of intracellular cholesterol. Our results indicate that binding to MPO is important for LDL to become modified and acquire proatherogenic properties. The peptide EQIQDDCTGDED, CP, ABAH, and SCN(-) can play the role of anti-atherogenic factors reducing the deleterious effect of catalytically active MPO on LDL and accumulation of cholesterol in macrophages.

Plasma autoantibodies against apolipoprotein B-100 peptide 210 in subclinical atherosclerosis.

OBJECTIVE: Experimental studies have suggested that autoimmunity is involved in atherosclerosis and provided evidence that both protective and pro-atherogenic immune responses exist. This concept has received support from small clinical studies implicating autoantibodies directed against apolipoprotein B-100 (apoB-100) in human atherosclerosis. We examined circulating autoantibodies directed against native and malondialdehyde (MDA)-modified epitope p210 of apoB-100 (IgG-p210nat and IgM-p210MDA) in relation to early atherosclerosis in a large, European longitudinal cohort study of healthy high-risk individuals. APPROACH AND RESULTS: IgG-p210nat and IgM-p210MDA were quantified in baseline plasma samples of 3430 participants in the IMPROVE study and related to composite and segment-specific measures of severity and rate of progression of carotid intima-media thickness (cIMT) determined at baseline and after 30 months. IgM-p210MDA autoantibody levels were independently related to several cIMT measures both in the common carotid artery and in the carotid bulb, including measures of cIMT progression, higher levels being associated with lower cIMT or slower cIMT progression. Consistent inverse relationships were also found between plasma levels of IgG-p210nat and baseline composite measures of cIMT. These associations disappeared when adjusting for established and emerging risk factors, and there were no associations with rate of cIMT progression besides in certain secondary stratified analyses. CONCLUSIONS: The present study provides further evidence of involvement of autoantibodies against native and MDA-modified apoB-100 peptide 210 in cardiovascular disease in humans and demonstrates that these associations are present already at a subclinical stage of the disease.

Protein-conjugated quantum dots interface: binding kinetics and label-free lipid detection.

We propose a label-free biosensor platform to investigate the binding kinetics using antigen-antibody interaction via electrochemical and surface plasmon resonance (SPR) techniques. The L-cysteine in situ capped cadmium sulfide (CdS; size < 7 nm) quantum dots (QDs) self-assembled on gold (Au) coated glass electrode have been covalently functionalized with apolipoprotein B-100 antibodies (AAB). This protein conjugated QDs-based electrode (AAB/CysCdS/Au) has been used to detect lipid (low density lipoprotein, LDL) biomolecules. The electrochemical impedimetric response of the AAB/CysCdS/Au biosensor shows higher sensitivity (32.8 kΩ µM(-1)/cm(2)) in the detection range, 5-120 mg/dL. Besides this, efforts have been made to investigate the kinetics of antigen-antibody interactions at the CysCdS surface. The label-free SPR response of AAB/CysCdS/Au biosensor exhibits highly specific interaction to protein (LDL) with association constant of 33.4 kM(-1) s(-1) indicating higher affinity toward LDL biomolecules and a dissociation constant of 0.896 ms(-1). The results of these studies prove the efficacy of the CysCdS-Au platform as a high throughput compact biosensing device for investigating biomolecular interactions.

A liposomal delivery system that targets liver endothelial cells based on a new peptide motif present in the ApoB-100 sequence.

Liver dysfunction is associated with a variety of liver diseases, including viral or alcoholic hepatitis, fibrosis, cirrhosis, and portal hypertension. A targeted drug delivery system would be very useful in the treatment of these diseases. We herein describe the development of a system comprised of a new peptide-lipid conjugate for the efficient delivery of molecules to LEC. The RLTRKRGLK sequence (3359-3367), which mediates the association of LDL with arterial CSPG and an LDL receptor, was utilized as a ligand for achieving this goal. The peptide modified PEG-LPs (RLTR-PEG-LPs) were efficiently taken up by primary liver endothelial cells (liver ECs) and other types of cells. In vivo biodistribution and confocal microscopy analysis showed that RLTR-PEG-LPs became widely accumulated in LECs within a short time. Distribution of RLTR-PEG-LPs was greatly reduced with a pretreatment of unlabeled RLTR-PEG-LPs, not cationic LPs, indicating that the sequence is important for LECs. The findings indicate that a reverse sequence of RLTR (KLGR) modified PEG-LPs (KLGR-PEG-LP) did the same pattern compared with RLTR-PEG-LPs, suggesting that the RKR or RXXR sequence might be essential for LECs targeting. Collectively RLTR-PEG-LPs and KLGR-PEG-LPs have the potential for delivering drugs to LECs.

Role of cysteine-protease CGHC motifs of ER-60, a protein disulfide isomerase, in hepatic apolipoprotein B100 degradation.

Apolipoprotein B100 (apoB), the structural component of very low density lipoproteins (VLDL), is susceptible to misfolding and subsequent degradation by several intracellular pathways. ER-60, which has been implicated in apoB degradation, is a protein disulfide isomerase (PDI) that forms or rearranges disulfide bonds in substrate proteins and also possesses cysteine protease activity. To determine which ER-60 function is important for apoB degradation, adenoviruses encoding wild-type human ER-60 or a mutant form of human ER-60 (C60A, C409A) that lacked cysteine protease activity were overexpressed in HepG2 cells. Overexpression of wild-type ER-60 in HepG2 cells promoted apoB degradation and impaired apoB secretion, but mutant ER-60 overexpression did not. In McArdle RH-7777 cells, VLDL secretion was markedly inhibited following overexpression of wild-type but not mutant ER-60, an effect that could be blocked by oleate treatment. Mutant ER-60 was not trapped on apoB as it was with the control substrate tapasin, suggesting that ER-60's role in apoB degradation is likely unrelated to its protein disulfide isomerase activity. Thus, ER-60 may participate in apoB degradation by acting as a cysteine protease. We postulate that apoB cleavage by ER-60 within the ER lumen could facilitate proteasomal degradation of the C-terminus of translocationally-arrested apoB.

A chemical approach for cell-specific targeting of nanomaterials: small-molecule-initiated misfolding of nanoparticle corona proteins.

A major challenge in nanomaterial science is to develop approaches that ensure that when administered in vivo, nanoparticles can be targeted to their requisite site of action. Herein we report the first approach that allows for cell-specific uptake of nanomaterials by a process involving reprogramming of the behavior of the ubiquitous protein corona of nanomaterials. Specifically, judicious surface modification of quantum dots with a small molecule that induces a protein-misfolding event in a component of the nanoparticle-associated protein corona renders the associated nanomaterials susceptible to cell-specific, receptor-mediated endocytosis. We see this chemical approach as a new and general method for exploiting the inescapable protein corona to target nanomaterials to specific cells.