Modification Of Apolipoprotein Research Articles

CIGB-258 Exerts Potent Anti-Inflammatory Activity against Carboxymethyllysine-Induced Acute Inflammation in Hyperlipidemic Zebrafish via the Protection of Apolipoprotein A-I.

Inflammation and atherosclerosis are intimately associated via the production of dysfunctional high-density lipoproteins (HDL) and modification of apolipoprotein (apo) A-I. A putative interaction between CIGB-258 and apoA-I was investigated to provide mechanistic insight into the protection of HDL. The protective activity of CIGB-258 was tested in the CML-mediated glycation of apoA-I. The in vivo anti-inflammatory efficacy was compared in paralyzed hyperlipidemic zebrafish and its embryo in the presence of CML. Treatment of CML induced greater glycation extent of HDL/apoA-I and proteolytic degradation of apoA-I. In the presence of CML, however, co-treatment of CIGB-258 inhibited the glycation of apoA-I and protected the degradation of apoA-I, exerting enhanced ferric ion reduction ability. Microinjection of CML (500 ng) into zebrafish embryos resulted in acute death with the lowest survivability with severe developmental defects with interleukin (IL)-6 production. Conversely, a co-injection of CIGB-258 or Tocilizumab produced the highest survivability with a normal development speed and morphology. In hyperlipidemic zebrafish, intraperitoneal injection of CML (500 μg) caused the complete loss of swimming ability and severe acute death with only 13% survivability 3 h post-injection. A co-injection of the CIGB-258 resulted in a 2.2-fold faster recovery of swimming ability than CML alone, with higher survivability of approximately 57%. These results suggest that CIGB-258 protected hyperlipidemic zebrafish from the acute neurotoxicity of CML. Histological analysis showed that the CIGB-258 group had 37% lower infiltration of neutrophils in hepatic tissue and 70% lower fatty liver changes than those of the CML-alone group. The CIGB-258 group exhibited the smallest IL-6 expression in the liver and the lowest blood triglyceride level. CIGB-258 displayed potent anti-inflammatory activity in hyperlipidemic zebrafish by inhibiting apoA-I glycation, promoting rapid recovery from the paralysis of CML toxicity and suppression of IL-6, and lowering fatty liver changes.

Isoform-specific modification of apolipoprotein E by 4-hydroxynonenal: protective role of apolipoprotein E3 against oxidative species.

High levels of 4-hydroxynonenal (HNE), arising from lipid peroxidation, and HNE-modified proteins have been identified in postmortem brains of ageing and Alzheimer's disease (AD) patients. The goal of this study is to understand the effect of HNE modification on the structure and function of recombinant apolipoprotein E3 (apoE3) and apolipoprotein E4 (apoE4), which play a critical role in brain cholesterol homeostasis. The two isoforms differ in a single amino acid at position 112: Cys in apoE3 and Arg in apoE4. Immunoblot with HNE-specific antibody indicates HNE modification of apoE3 and apoE4 with a major band at ~ 36 kDa, while LC-MS/MS revealed Michael addition at His140 (60-70% abundance) and His299 (3-5% abundance) in apoE3 and apoE4, and Cys112 adduct in apoE3 (75% abundance). Circular dichroism spectroscopy revealed no major differences in the overall secondary structure or helical content between unmodified and HNE-modified apoE. HNE modification did not affect their ability to promote cholesterol efflux from J774.1 macrophages. However, it led to a 3-fold decrease in their ability to bind lipids and 25-50% decrease in the ability of cerebral cortex endothelial cells to uptake lipoproteins bearing HNE-modified HNE-apoE3 or HNE-apoE4 as noted by fluorescence microscopy and flow cytometry. Taken together, the data indicate that HNE modification impairs lipid binding and cellular uptake of both isoforms, and that apoE3, bearing a Cys, offers a protective role by sequestering lipid peroxidation products that would otherwise cause indiscriminate damage to biomolecules. ApoE4, lacking Cys, is unable to protect against oxidative damage that is commensurate with ageing.

487-P: Posttranslational Modifications of Apolipoproteins C-I, C-II, and C-III and Peripheral Artery Disease in Multiethnic Study of Atherosclerosis (MESA)

Apolipoproteins (apo) C-I, C-II and C-III in circulation are in several post-translational forms with distinct effects on lipoprotein metabolism. ApoC-I and apoC-II exist as dominant native and minor truncated (C-I’; C-II’) forms; apoC-III appears as unglycosylated (C-III0a) , or glycosylated with zero (C-III0b) , one (C-III1, most abundant) or two (C-III2) sialic acids. The association of these proteoforms with peripheral artery disease (PAD) in a general population is unstudied. Total apoC-I, C-II and C-III concentrations and relative amounts of their proteoforms were measured in baseline plasma of 5,681 MESA participants. The ankle-brachial index (ABI) was assessed at baseline, 3 and years. Prevalent PAD was defined as baseline ABI ≤ 0.9. Incident PAD was follow-up ABI ≤ 0.9 or a hospital-based diagnosis of PAD to include revascularization. After adjustment for baseline characteristics, lipid lowering therapy and plasma lipids, low baseline ABI was associated with low C-III0b to C-III1 ratio (C-III0b/III1, p=0.01) and high C-III2 to C-III1 ratio (C-III2/III1, p<0.01) . Prevalent PAD (n=226) was only associated with C-III2/III1 (OR 1.16, 95% CI: 1.01-1.33, p=0.03) . Lower ABI at follow-up was associated with lower C-III0b/III1 and higher C-III2/III1 at baseline (p<0.both) . Incident PAD (n=244) was associated with C-III2/III1 (HR 1.22, 1.06-1.41, p=0.01) . Total apoC-I, C-II and C-III concentrations, or apoC-I and C-II truncations were not associated with ABI at baseline and follow-up, or with cross-sectional and prospective PAD determinations. ApoC-I and apoC-II and their truncations do not appear linked with PAD. In contrast, we find different associations of apoC-III0b and apoC-III2 with prevalent and incident PAD that are independent of other risk factors, including plasma lipids. Our data provide further support that proteoforms may have unique properties not captured by “total” apolipoprotein measures. Disclosure P.Reaven: Research Support; AstraZeneca, Dexcom, Inc. J.Koska: None. S.Hansen: None. Y.Hu: None. J.Furtado: Research Support; Eli Lilly and Company, Pfizer Inc. S.Sinari: None. D.Billheimer: None. D.Nedelkov: None. M.Budoff: Research Support; Amarin Corporation, Novo Nordisk, Speaker's Bureau; Boehringer Ingelheim International GmbH, Lilly. R.Mcclelland: None. Funding National Institutes of Health (R01-HL138969)

Redox equilibrium of serum apolipoprotein E3: a buffering effect of disulfide-linked complexes against oxidative stress on apolipoprotein E3–containing lipoproteins

Reversible redox modification of cysteine thiols is crucial for protecting proteins from irreversible detrimental change. However, the physiological significance of the redox modification of apolipoprotein (apo) E is unclear. Here, we hypothesized that the disulfide-linked complexes of apoE3 corresponding to the representative reversible-modified apoE3 play a protective role against oxidative stress. The effects of disulfide bond formation on oxidative stress on apoE3 were evaluated with a band-shift assay. Maleimide-labeled apoE3 and unlabeled apoE3 were defined as the reduced (r)-apoE3 and non-reduced (nr)-apoE3 forms, respectively. Hydrogen peroxide-induced oxidation decreased for reduced-form apoE (r-apoE3) but increased for nr-apoE3. Induction of apoE3-AII complex formation with excess of apoAII markedly suppressed the oxidative stress-induced increase in nr-apoE3 (P<0.001) and enhanced homodimer formation. The apoE3-AII complex was more dominant in high-density lipoprotein (HDL) than in very low-density lipoprotein. Under oxidative stress, HDL showed a significant decrease, rather than an increase, in nr-apoE3 levels with a concomitant significant increase in apoE3-AII levels (P<0.005). This finding suggests that the majority of nr-apoE3 in HDL exists in a reversible oxidized form. The apoE3-AII complex, formed from the reversible oxidized apoE3, is beneficial for maintaining the redox equilibrium of apoE3 by preventing the modification of apoE3 to its irreversible oxidized form. The apoE3-AII complex may be possibly implicated in the pathophysiology of various apoE-related diseases.

Increased electronegativity of high-density lipoprotein in uremia patients impairs its functional properties and is associated with the risk of coronary artery disease

Background and aimsUremia patients have impaired high-density lipoprotein (HDL) function and a high risk of coronary artery disease (CAD). Increased lipoprotein electronegativity can compromise lipoprotein function, but the effect of increased HDL electronegativity on HDL function and its association with CAD in uremia patient are not clear.We aimed to assess HDL electronegativity and various properties of HDL in uremia patients and investigate whether electronegative HDL is a risk factor for CAD in these individuals. MethodsHDL from 60 uremia patients and 43 healthy controls was separated into 5 subfractions (H1H5) with increasing electronegativity by using anion-exchange chromatography. Lipoprotein content was analyzed by gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry. HDL anti-oxidant, anti-apoptosis and cholesterol efflux activities were examined by fluorescence-based assays. ResultsThe percentage of H5 HDL (H5%) was significantly higher in uremia patients than in controls (p < 0.001). The concentration of apolipoprotein (Apo) AI was lower and apolipoprotein modifications were more prevalent in uremia HDL subfractions than in control HDL subfractions. Carbamylation of ApoAI and ApoCIII was increased in more electronegative HDL subfractions from uremia patients. Anti-oxidant activity, anti-apoptotic activity, and cholesterol efflux capability were reduced in HDL subfractions from uremia patients when compared with control HDL subfractions. Multiple logistic regression analysis showed that H5% was associated with CAD risk in uremia patients. ConclusionsIn HDL of uremia patients, increased electronegativity is accompanied by compositional changes and impaired function. Our findings indicate that increased H5% is associated with increased CAD risk in uremia patients.

Abstract 16: Amyloidogenic Modifications of Apolipoprotein A-I Promote a Strong Pro-inflammatory Response in Macrophages, but Formation of Amyloid Fibrils Abrogates the Pro-inflammatory Effect

Inflammation is central to atherosclerosis, as inactivation of inflammation pathways strongly reduces atherosclerosis progression. Particulate matter, such as cholesterol crystals and amyloid materials, promotes release of the potent pro-inflammatory cytokine IL-1β in macrophages. Recently, it has been demonstrated that soluble substances that are precursors of particulate matter, such as free cholesterol, oxidized LDL and amyloidogenic peptides (i.e. amyloid-β, IAPP), can also induce an inflammatory response. In this study, we investigated the interplay between oxidation of apolipoprotein A-I (apoA-I), amyloid formation, and the inflammatory response of macrophages. We previously reported that oxidation of apoA-I methionines (MetO-ApoA-I) promotes protein aggregation and formation of amyloid fibrils when MetO-ApoA-I is incubated at pH 6.0. In contrast, at physiological pH, MetO-ApoA-I remains soluble and perfectly functional, as it promotes cholesterol efflux from macrophages with the same efficiency of intact-ApoA-I. However, upon incubation of mouse bone marrow derived macrophages with soluble pre-fibrillar MetO-ApoA-I, levels of pro-IL-1β synthesis were more than 2-fold higher than those induced by intact-ApoA-I. In contrast, amyloid fibrils produced by MetO-ApoA-I did not increase the levels of pro-IL-1β synthesis compared to intact-ApoA-I. Furthermore, the pro-inflammatory effect was not observed in macrophages derived from MyD88/TRIF knock-out mice, indicating that the response is membrane TLR receptors-dependent. In contrast, the &gt;2-fold increase (MetO-ApoA-I vs. intact-apoA-I) in pro-IL-1β synthesis was maintained in macrophages derived from CD36 knock-out mice. This observation suggests that the signaling pathway is not exclusively dependent on the TLR2/TLR6/CD36 membrane complex. Thus, in atherosclerotic lesions, oxidized apoA-I species that are amyloidogenic, but otherwise functional, may induce a pro-inflammatory response in macrophages. Amyloid fibril formation in contrast, could reduce, rather than exacerbate, the inflammatory burden produced by these pro-inflammatory apoA-I species by sequestering them in the form of inert amyloids.

Abstract 14: Characterization of the Oxidized Phospholipid Modification of Apolipoprotein(a) Kringle KIV10: Insights Into the Site of OxPC Addition

Elevated plasma levels of lipoprotein(a) (Lp(a)) are an independent and causal risk factor for coronary heart disease and aortic valve stenosis. Lp(a) consists of a low density lipoprotein (LDL)-like particle covalently linked to the unique glycoprotein apolipoprotein(a) (apo(a)). We have shown that apo(a) contains a covalent oxidized phosphocholine (oxPC) adduct on the KIV 10 domain, and that perturbation of the strong lysine binding site (LBS) in this kringle results in a lack of covalent oxPC addition. We have implicated this modification in proinflammatory processes, such as the ability of apo(a) to induce interleukin-8 expression in macrophages. Apo(a) from Old World monkeys and apes lacks covalent oxPC modification and contains mutations in KIV 10 , some of which impact the LBS. To identify the amino acids in human apo(a) that are covalently modified by oxPC, we mutagenized a KIV 10 KV di-kringle apo(a) that contains covalent oxPC modification. We mutated to alanine all residues that are either substituted in primate apo(a) or that can act as acceptors for covalent oxPC addition. The resulting variants were then subjected to immunoblotting analysis with E06, an IgM antibody that binds to oxPC, and assessment of lysine-binding ability using affinity chromatography. Mutation of His33 to Ala abolishes oxPC modification of apo(a) while retaining the lysine binding ability of KIV 10 . Thus, we have identified the long-sought location of the oxPC modification of apo(a). Molecular dynamic simulations of the oxPC-deficient mutant show no gross changes in the structure of KIV 10 , in contrast to mutations that abolish both lysine binding ability and oxPC addition that result in a collapsed lysine binding pocket. Using mass spectrometry, we have also identified noncovalently-associated oxPC present on apo(a). Interestingly, the abundance of these species appears to be reduced in the apo(a) variant containing a mutation in the strong lysine binding site in KIV 10 . Our findings allow the ability to assess the contributions to pathogenesis of the strong lysine binding site and oxPC modification of apo(a) kringle IV type 10 independently, and thus will enhance our understanding of the mechanisms by which Lp(a) contributes to atherothrombotic diseases.

Abstract 389: Covalent Modifications of Apolipoproteins in High Density Lipoprotein by Oxidized Phospholipids Impair the Cholesterol Efflux Function of High Density Lipoprotein and are Detected in Hyperlipidemic Plasma

Oxidized phospholipids (oxPLs) accumulate at sites of oxidative stress and contribute significantly to atherosclerosis and thrombosis. Most oxPLs have electrophilic substituents and are highly likely to form covalent adducts with proteins, thus compromising protein function. Detection of covalent interactions between oxPLs and proteins could provide important information regarding the type of proteins preferentially modified by oxPLs. However, to date, such studies are extremely limited due to significant technical challenges. We now carry out systematic studies on the protein adduction by oxPLs formed in murine and human plasma. Plasma samples were exposed to a physiologically relevant myeloperoxidase/H 2 O 2 /NO 2 – oxidizing system of phagocytes. Protein adduction by the oxPLs generated in plasma was assessed using LC-MS/MS after tryptic digestion and peptide enrichment using a novel method that we developed. We found that HDL apolipoproteins are the major targets of modification by oxPLs in both murine and human plasma. For apoA-I, the most abundant apolipoprotein in HDL, the major modification sites of oxPLs were located in the region (AA144-186), which is critical for the ABCA1 mediated cholesterol efflux to HDL. We further demonstrated that human apoA-I was also heavily crosslinked by specific oxPLs via histidine and lysine residues located in the region (AA144-186) with apoA-I, or other apolipoproteins, including apoA-II, apoA-IV and apoC-I. In vitro experiments demonstrated that oxPLs modification on lipid free apoA-I or nascent HDL (HDL3) dramatically impairs their function as cholesterol efflux mediators. Using hyperlipidemic LDLr-/- mice, we detected a crosslink adduct of apoA-II with apoE by oxPL in murine plasma. To the best of our knowledge, this is the first report for the detection of endogenous protein adducts with oxPLs.

Cigarette smoke exposure impairs reverse cholesterol transport which can be minimized by treatment of hydrogen-saturated saline.

BackgroundCigarette smoke (CS) exposure impaired plasma lipid profiles by modification of apolipoproteins. Hydrogen (H2) has been proved effective on reducing oxidative stress or improving HDL functionalities in animal models or metabolic syndrome volunteers. This study was undertaken to explore the effects of CS exposure on reverse cholesterol transport (RCT) and the antioxidative effects of H2 treatment against CS exposure in mice transgenic for human cholesteryl ester transfer protein (CETP).Methods[3H]-cholesterol-laden macrophages were injected intraperitoneally into mice, and the samples of blood, bile, liver, and feces were collected for radioactivity determination to evaluate RCT. [3H]-cholesterol-laden macrophages were incubated with HDL isolated from different groups of mice, and the samples of cell medium supernatants were collected for evaluating the HDL functionality to elicit cholesterol efflux.ResultsCS exposure significantly decreased plasma HDL cholesterol level (HDL-C) by 22 % and increased LDL cholesterol level (LDL-C) by 21 % compared with the control group (p < 0.05, p < 0.01), while H2 treatment significantly improved the CS-impaired levels of TC, LDL-C and HDL-C by 10, 27 and 31 %, respectively, compared with the CS group (p < 0.05, p < 0.01 and p < 0.05). Besides, CS exposure significantly decreased [3H] tracer concentrations in liver, bile and feces by 17, 35 and 48 %, respectively, compared with the control group (p < 0.05 for liver and feces), while H2 treatment significantly improved them by 21, 72 % and 89 %, respectively, compared with the CS group (all p < 0.05). Furthermore, CS exposure significantly decreased the HDL functionality to elicit cholesterol efflux by 26 % (p < 0.05), while H2 treatment also improved it by 32 % (p < 0.05). We did not find any significant alterations in protein expressions of RCT involved genes.ConclusionsThese findings provided direct evidence supporting the notion that CS exposure in vivo impairs plasma lipid profiles, HDL functionalities and macrophage-to-feces RCT pathway in CETP transgenic mice, all of which can be minimized by treatment of H2-saturated saline.

The application of multiple reaction monitoring to assess ApoA-I methionine oxidations in diabetes and cardiovascular disease

The oxidative modification of apolipoprotein A-I’s methionine148 (M148) is associated with defective HDL function in vitro. Multiple reaction monitoring (MRM) is a mass spectrometric technique that can be used to quantitate post-translational modifications. In this study, we developed an MRM assay to monitor the abundance ratio of the peptide containing oxidized M148 to the native peptide in ApoA-I. Measurement of the oxidized-to-unoxidized-M148 ratio was reproducible (CV < 5%). The extent of methionine M148 oxidation in the HDL of healthy controls, and type 2 diabetic participants with and without prior cardiovascular events (CVD) were then examined. The results suggest a significant increase in the relative ratio of the peptide containing oxidized M148 to the unmodified peptide in the HDL of participants with diabetes and CVD (p < 0.001), compared to participants without CVD. Monitoring the abundance ratio of the peptides containing oxidized and unoxidized M148 by MRM provides a means of examining the relationship between M148 oxidation and vascular complications in CVD.

Analysis of Covalent Modifications of Proteins by Oxidized Phospholipids Using a Novel Method of Peptide Enrichment

Free radical-induced oxidation of phospholipids contributes significantly to pathologies associated with inflammation and oxidative stress. Detection of covalent interaction between oxidized phospholipids (oxPL) and proteins by LC-MS/MS could provide valuable information about the molecular mechanisms of oxPL effects. However, such studies are very limited because of significant challenges in detection of the comparatively low levels of oxPL-protein adducts in complex biological systems. Current approaches have several limitations, most important of which is the inability to detect protein modifications by naturally occurring oxPL. We now report, for the first time, an enrichment method that can be applied to the global analysis of protein adducts with various naturally occurring oxPL in relevant biological systems. This method exploits intrinsic properties of peptides modified by oxPL, allowing highly efficient enrichment of oxPL-modified peptides from biological samples. Very low levels of oxPL-protein adducts (<2 ppm) were detected using this enrichment method in combination with LC-MS/MS. We applied the method to several model systems, including oxidation of high density lipoprotein (HDL) and interaction of human platelets with a specific oxPL, and demonstrated its extremely high efficiency and productivity. We report multiple new modifications of apolipoproteins in HDL and proteins in human platelets.

Apolipoproteins in High-Density Lipoproteins (HDL) and their modifications determine the Sphingosine 1-Phosphate (S1P) content of HDL: effects on the HDL-mediated, S1P-dependent signaling

Purpose: The bioactive sphingolipid sphingosine 1-phosphate (S1P) binds in plasma mainly to high-density lipoproteins (HDL) via apolipoprotein M (apoM) and mediates many of the atheroprotective and cardioprotective properties of HDL. Recently, we demonstrated that the S1P-content of plasma and HDL is reduced in patients with coronary artery disease (CAD). In the current study, we tested the hypothesis that the reduction of S1P in CAD-HDL is due to modifications of apolipoproteins in HDL. In addition, we tested whether the extent of cellular responses upon stimulation with HDL was related to the S1P content of HDL. Methods: HDL were isolated by density gradient ultracentrifugation from plasma of patients with CAD (n=65) and of controls (n=68). The contents of apoM, apoAI and S1P were detected by western blotting, ELISA and mass spectroscopy, respectively. The uptake capacity of HDL for S1P was measured in native and oxidized HDL as well as in the subfractions HDL2 and HDL3. Induction of the phosphorylation of p42/44 Erks upon stimulation with HDL was determined in S1P-receptor 1-overexpressing CHO-cells. The same assays were performed in HDL isolated from plasma of apoM-knock out, apoAI knock-out, and apoAI-transgenic mice. Results: While the S1P content was lower in CAD-HDL, the contents of apoM and apoAI were similar in CAD-HDL and control HDL. Oxidation of HDL reduced their baseline S1P content by 44% and their uptake capacity for S1P by 69%. HDL3 had less S1P than HDL2 not only at baseline (122 pmol/mg of protein [102-171] vs. 225 pmol/mg of protein [216-448], P=0.004) but also after loading HDL with S1P (1510 pmol/mg of protein [930-1990] vs. 5732 pmol/mg of protein [5092-7252], P=0.004). HDL isolated from apoM-deficient and apoAI-transgenic mice had a significantly reduced content of S1P (6.3% and 20.2%, respectively). In addition, intracellular phosphorylation of p42/44 Erks after stimulation with apoM-deficient HDL was greatly diminished. An enormous capacity of HDL to uptake S1P was found (20 fold of baseline). Loading apoM-deficient HDL with S1P normalized the defective activation of p42/44 Erks. HDL mediated cell signaling was completely blocked by a S1P neutralizing antibody. Conclusion: Changes in the apolipoprotein composition of HDL, posttranslational modifications of apolipoproteins, or the disarranged ratio of HDL2/HDL3 in patients with CAD might be causes for the reduction of S1P in plasma and in HDL in CAD. As the content of S1P in HDL seems to determine the extent of HDL-mediated effects, quality and function of HDL might be improved by targeting their S1P content.

Abstract 103: Mass Spectral Analysis of Oxidative Stress Modification of Apolipoprotein E by Acrolein

Apolipoprotein E (apoE), an anti-atherogenic apolipoprotein, plays a significant role in the metabolism of lipoproteins. It lowers plasma lipid levels by acting as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins. ApoE mediates this function via essential lysine residues that interact with the LDLr. Preliminary studies from our lab showed that rats exposed to environmental tobacco smoke displayed oxidative modification of apoE by acrolein (a highly reactive unsaturated aldehyde) and dissociation of lipoprotein-bound apoE; however, the details regarding the modification were not determined at the molecular level. The objectives of this project are to identify the specific sites of modification and the effect of modification on the structure and function of recombinant rat apoE. Acrolein modification was verified by Western blot analysis using an acrolein-lysine specific antibody. Matrix Assisted Laser Desorption Ionization-Time of Flight/Time of Flight Mass Spectrometry (MALDI-TOF/TOF MS) analysis identified Lys64, 67, 68, 138 &amp; 234 as likely modification sites by acrolein. Guanidine HCl-induced denaturation studies indicated that the overall fold of the modified protein was significantly altered as revealed by circular dichroism spectroscopy. Modified apoE also demonstrated a decrease in heparin binding affinity and lipid binding ability. The LDLr binding ability of modified apoE was significantly impaired. Loss of LDLr binding ability is attributed to acrolein modification of Lys138, an essential residue located on helix 4, that is directly involved in interaction with the LDLr as seen for human apoE3. Overall, we conclude that oxidative modification by acrolein disrupts the structural and functional integrity of apoE, which is likely to affect its role in maintaining plasma cholesterol homeostasis. Our data provide a molecular basis for the potential role of oxidative stress- (due to environmental factors or aging) mediated modification of apoE in altering lipoprotein metabolism, with direct implications in cardiovascular disease.

Altered Plasma Apolipoprotein Modifications in Patients with Pancreatic Cancer: Protein Characterization and Multi-Institutional Validation

BackgroundAmong the more common human malignancies, invasive ductal carcinoma of the pancreas has the worst prognosis. The poor outcome seems to be attributable to difficulty in early detection.MethodsWe compared the plasma protein profiles of 112 pancreatic cancer patients with those of 103 sex- and age-matched healthy controls (Cohort 1) using a newly developed matrix-assisted laser desorption/ionization (oMALDI) QqTOF (quadrupole time-of-flight) mass spectrometry (MS) system.ResultsWe found that hemi-truncated apolipoprotein AII dimer (ApoAII-2; 17252 m/z), unglycosylated apolipoprotein CIII (ApoCIII-0; 8766 m/z), and their summed value were significantly decreased in the pancreatic cancer patients [P = 1.36×10−21, P = 4.35×10−14, and P = 1.83×10−24 (Mann-Whitney U-test); area-under-curve values of 0.877, 0.798, and 0.903, respectively]. The significance was further validated in a total of 1099 plasma/serum samples, consisting of 2 retrospective cohorts [Cohort 2 (n = 103) and Cohort 3 (n = 163)] and a prospective cohort [Cohort 4 (n = 833)] collected from 8 medical institutions in Japan and Germany.ConclusionsWe have constructed a robust quantitative MS profiling system and used it to validate alterations of modified apolipoproteins in multiple cohorts of patients with pancreatic cancer.

Effects of Oxidation on Structural Stability and Remodeling of Human Very Low Density Lipoprotein

Very low density lipoproteins (VLDL) are triglyceride-rich precursors of low-density lipoproteins (LDL) and a risk factor for atherosclerosis. The effects of oxidation on VLDL metabolism may be pro- or antiatherogenic. To understand the underlying biophysical basis, we determined the effects of copper (that preferentially oxidizes lipids) and hypochlorite (that preferentially oxidizes proteins) on the heat-induced VLDL remodeling. This remodeling involves VLDL fusion, rupture, and fission of apoE-containing high-density lipoprotein- (HDL-) like particles; HDL with similar size, density, and protein composition are formed upon VLDL remodeling by lipoprotein lipase, a key enzyme in triglyceride metabolism. Circular dichroism, turbidity, and electron microscopy show that mild oxidation promotes VLDL fusion and rupture, while advanced oxidation hampers these reactions. VLDL destabilization upon moderate oxidation results, in part, from the exchangeable apolipoprotein modifications, including proteolysis and limited cross-linking. VLDL stabilization against fusion and rupture upon advanced oxidation probably results from massive protein cross-linking on the particle surface. Electron microscopy and gel electrophoresis reveal that oxidation promotes fission of apoE-containing HDL-size particles; hydrolysis of apolar core lipids probably contributes to this effect. Copper and hypochlorite have similar effects on VLDL remodeling, suggesting that these effects may be produced by other oxidants. In summary, moderate oxidation that encompasses in vivo conditions destabilizes VLDL and promotes fission of HDL-size particles. Consequently, mild oxidation may be synergistic with lipoprotein lipase reaction and, hence, may help to accelerate VLDL metabolism.

Sphingosine kinase regulates oxidized low density lipoprotein-mediated calcium oscillations and macrophage survival

We recently reported that oxidized LDL (oxLDL) induces an oscillatory increase in intracellular calcium ([Ca(2+)](i)) levels in macrophages. Furthermore, we have shown that these [Ca(2+)](i) oscillations mediate oxLDL's ability to inhibit macrophage apoptosis in response to growth factor deprivation. However, the signal transduction pathways by which oxLDL induces [Ca(2+)](i) oscillations have not been elucidated. In this study, we show that these oscillations are mediated in part by intracellular mechanisms, as depleting extracellular Ca(2+) did not completely abolish the effect. Inhibiting sarco-endoplasmic reticulum ATPase (SERCA) completely blocked [Ca(2+)](i) oscillations, suggesting a role for Ca(2+) reuptake by the ER. The addition of oxLDL resulted in an almost immediate activation of sphingosine kinase (SK), which can increase sphingosine-1-phosphate (S1P) levels by phosphorylating sphingosine. Moreover, S1P was shown to be as effective as oxLDL in blocking macrophage apoptosis and producing [Ca(2+)](i) oscillations. This suggests that the mechanism in which oxLDL generates [Ca(2+)](i) oscillations may be 1) activation of SK, 2) SK-mediated increase in S1P levels, 3) S1P-mediated Ca(2+) release from intracellular stores, and 4) SERCA-mediated Ca(2+) reuptake back into the ER.

Evalution of in vitro effect of flavonoids on human low-density lipoprotein carbamylation.

The non-enzymatic carbamylation of low density lipoprotein (LDL) is a naturally occurring chemical modification of apolipoprotein B as a result of condensation between lysine residues and cyanate derived from urea. Carbamylated LDL is poorly recognized by LDL receptors and initiates different processes that can be considered proatherogenic. Thus, LDL carbamylation may contribute to the increased risk of atherosclerosis in patients with kidney failure. The objective of this study was to investigate in vitro effects of flavonoids on LDL carbamylation. LDL was isolated from plasma using ultracentrifuge technique with a single step discontinuous gradient. Then, cyanate was added to LDL and LDL carbamylation level was estimated in absence and presence of flavonoids by a colorimetric method at 530 nm. The results of this study showed that a number of flavonoids including rutin, catechin, morin, myricetin, kaempferol, taxifolin, luteolin, naringin and quercetin decreased LDL carbamylation in a dose dependent manner. Also, it was demonstrated that these nutrients decreased electrophoretic mobility of carbamylated LDL. Based on the results obtained in this study, it is suggested that flavonoids are able to inhibit LDL carbamylation (probably by scavenging cyanate ions) and thus, may have a role in ameliorating atherosclerotic risk of patients with kidney failure.

LOX-1 augments oxLDL uptake by lysoPC-stimulated murine macrophages but is not required for oxLDL clearance from plasma

Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to define the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of 125I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-inflammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.

HDL, lipid peroxidation, and atherosclerosis

Genetic, clinical, and pharmacological studies implicate elevated levels of LDL in the pathogenesis of atherosclerotic vascular disease, the leading cause of death in industrialized societies (1). Paradoxically, native LDL fails to exert potentially atherogenic effects in vitro, suggesting that it must be modified to promote vascular disease. Indeed, many lines of evidence support the LDL oxidation hypothesis, which suggests that oxidative damage to LDL is one important mechanism for rendering lipoproteins atherogenic (as reviewed in Refs. 2, 3).

Protective Effect of Magnoflorine Isolated from Coptidis Rhizoma on Cu2+-Induced Oxidation of Human Low Density Lipoprotein

The aim of this study was to investigate the antioxidant activity of magnoflorine, an alkaloid isolated from Coptidis Rhizoma, against the oxidation of native low density lipoprotein (LDL) and modified LDL. Magnoflorine was found to inhibit the copper-mediated (Cu2+) oxidation of LDL, as well as of glycated and glycoxidated LDL by increasing the lag time of conjugated diene formation and preventing the generation of thiobarbituric acid reactive substances (TBARS). In addition, the results from the fluorescence emission spectra of tryptophan (Trp) supported that the antioxidant activity of magnoflorine could be associated with the protective effect on the structural modification of apolipoprotein B (apoB) required for LDL oxidation. These results suggest that magnoflorine may be useful for preventing the oxidation of various LDL forms.