Anti-ApoA-1 IgGs in Familial Hypercholesterolemia Display Paradoxical Associations with Lipid Profile and Promote Foam Cell Formation

Anti-Apolipoprotein A-1 autoantibodies (anti-ApoA-1 IgG) promote atherogenesis via innate immune receptors, and may impair cellular cholesterol homeostasis (CH). We explored the presence of anti-ApoA-1 IgG in children (5-15 years old) with or without familial hypercholesterolemia (FH), analyzing their association with lipid profiles, and studied their in vitro effects on foam cell formation, gene regulation, and their functional impact on cholesterol passive diffusion (PD)

Studies of CD36 interacting with fatty acids, oxidized low-density lipoprotein, and the cellular plasma membrane

The glycoprotein CD36 is expressed in the plasma membrane (PM) of many cell types that surround or contact arteries, including macrophages, myocytes, and endothelial cells. CD36 binds oxidized low density lipoprotein (oxLDL), which promotes atherosclerosis, and fatty acids (FA), which promotes their cellular uptake. To gain insights into the molecular mechanisms of uptake, HEK293 cells expressing CD36 were studied by cell biological and fluorescence methods. To test our hypothesis that the PM is not an impermeable barrier to FA and that FA move into cells by diffusion via their uncharged form, we first applied biophysical fluorescence spectroscopy to directly measure transmembrane FA movement and membrane fluidity. Expression of CD36 in HEK293 cells did not increase either transport across the PM or the fluidity of the PM compared to HEK293 cells without CD36; however, CD36 enhanced intracellular FA esterification. Furthermore, the widely used “inhibitors” of FA transport did not alter either the rapid FA transmembrane diffusion in HEK293 cells or diffusion in control experiments with protein-free phospholipid bilayers. To gain new insights into the physiological relevance of FA binding to CD36, we applied surface plasmon resonance (SPR) to quantify FA and oxLDL binding to the ectodomain of CD36. Structurally distinct FA [saturated, monounsaturated (cis and trans), polyunsaturated, ω-3, ω-6, and oxidized FA] were pulsed in a solubilized form (bound to methyl-β-cyclodextrin) across SPR channels, generating real-time association and dissociation binding curves. With the exception of the oxidized FA hydroxyoctadecadienoic acid (HODE), all FA tested bound to CD36 with rapid association and dissociation kinetics similar to human serum albumin. In addition, FA increased oxLDL binding to CD36. To investigate whether FA affect CD36-mediated oxLDL uptake in live cells, we monitored fluorescent oxLDL (Dii-oxLDL) uptake using confocal microscopy. Addition of exogenous FA to serum-free media enhanced dose-dependent oxLDL uptake. Exceptions were ω-3 FA, which bound to CD36, and HODE, which did not bind to CD36, demonstrating FA structure-specific effects on a major function of CD36 and a new mechanistic link between atherosclerosis and high levels of FA in obese and Type-II diabetic individuals

Biophysical studies of motions and interactions of membrane proteins

2011 Fall.Includes bibliographical references.We have utilized a variety of biophysical techniques to quantitatively examine the motions and interactions of transmembrane proteins on living cells at the single-molecule level. These include both widefield and confocal optical microscopic methods such as single particle tracking, Förster resonance energy transfer and ratiometric imaging of phase-sensitive probes of lipid order, together with spectroscopic fluctuation methods such as fluorescence correlation spectroscopy and photon counting histogram analysis. Our studies indicate that; 1. Luteinizing hormone receptors on CHO cells and KGN human granulosa cells exhibit restricted lateral diffusion and increased self-association after exposure to human chorianic gonadotropin; 2. In addition to insulin receptor and IGF1 receptor homodimers, rat basophilic leukemia 2H3 cells express significant levels of insulin receptor-IGF1 receptor heterodimers; 3. Clustering of insulin receptors after exposure to insulin on rat basophilic leukemia 2H3 cells is affected by disruption of actin-filaments but not by extraction of membrane cholesterol; 4. Chromium Picolinate and Bis(maltolato)oxovandium(IV) both restrict the lateral diffusion of insulin receptors on rat basophilic leukemia cells and; 5. Individual FcE receptors on rat basophilic leukemia cells exhibit orientation fluctuations on millisecond timescales

POLYCHLORINATED BIPHENYL-INDUCED ENDOTHELIAL CELL DYSFUNCTION AND ITS MODULATION BY DIETARY LIPIDS

Cardiovascular diseases are the number one cause of death in Western societies. Endothelial dysfunction is an early event in the pathology of atherosclerosis, which is an underlying cause in the majority of cardiovascular events. Exposure to persistent environmental pollutants, such as polychlorinated biphenyls (PCBs), is a risk factor for the development of atherosclerosis. First, we tested a hypothesis that coplanar PCBs, dioxin-like chemicals with affinity for aryl hydrocarbon receptor (AhR), can stimulate up-regulation of monocyte chemoattractant protein-1 (MCP-1), an endothelium-derived chemokine that attracts monocytes into sub-endothelial space in early stages of atherosclerosis. Coplanar PCBs 77 and 126 increased expression of MCP-1 in endothelial cells, and this effect was dependent on activation of AhR and increased levels of cytochrome P450 monoxygenases. Subsequent rise in the levels of reactive oxygen species (ROS) led to a downstream stimulation of redox-sensitive kinases and transcription factors. Lipid rafts, and particularly caveolae, are enriched in endothelial cells, and down-regulation of caveolin-1, a key structural protein of caveolae, decreases the progression of atherosclerosis. Studies using deletion of caveolin-1 in vitro and in vivo demonstrated that intact caveolae were required for up-regulation of MCP-1 and pro-inflammatory interleukin-6 (IL-6) by PCB77. Nutrition can modulate adverse outcomes of human exposure to environmental chemicals. Fish oil-derived long-chain omega-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6ω-3), can alleviate inflammatory responses and the risk of cardiovascular disease. Cyclopentenone metabolites produced by oxidation of DHA contribute to these protective effects. Endothelial cells were pre-treated with oxidized DHA (oxDHA), prepared by incubation of the fatty acid with a free radical generator. Subsequent up-regulation of MCP-1 by coplanar PCB77 was markedly reduced. DHA-derived cyclopentenones increased nuclear translocation and DNA binding of a transcription factor NF-E2-related factor-2 (Nrf2), as well as expression levels of its target, antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO1). This stimulation of antioxidant responses prevented ROS production and inflammatory responses induced by PCB77. These data support the concept that nutrition prevents toxicity caused by environmental pollutants; thus, nutrition and can be a sensible approach to alleviate chronic pathologies associated with these chemicals

Vitamin E supplementation and atherosclerosis : epidemiological studies in elderly and smokers

The antioxidant vitamin E may have beneficial effects on several indicators of human health. We studied the impact on atherosclerosis, immune response and total mortality in smokers and elderly people, who are at risk for increased oxidative stress. Vitamin E may exert its effect on atherosclerosis by protecting low density lipoprotein (LDL) against lipid peroxidation. Moreover, lipid peroxidation may also negatively influence the immune response. In addition to its antioxidant function, vitamin E may be beneficial through effects on cellular function, e.g., by preserving endothelium-dependent vaso-relaxation or decreasing cytokine production.Observational studies . In a cross-sectional study among 158 male lifelong smokers aged 50 to 75 years, adjusted vitamin E intake and plasma levels were not associated with intima media wall thickness (IMT) of the common carotid artery. IMT ,which is a marker for atherosclerosis, was measured non-invasively by using the B-mode ultrasound technique. In a prospective study among 638 independently living elderly aged 65 to 85, no significant association was observed between cholesterol adjusted serum levels of vitamin E and 7.2 years total mortality. (Hazard ratio for lowest tertile vs. highest of 1.11, 95% confidence interval 0.74 to 1.65).Intervention trials. In a randomized placebo-controlled double-blind trial among 218 lifelong male smokers, 400 IU (364 mg) vitamin E was administered daily for two years. A non significant (p=0.34) reduced progression of carotid IMT by 47% was observed compared to a significant spontaneous progression in the placebo group of 0.030 mm (p=0.006). Results were adjusted for initial IMT values and traditional CVD risk factors. Vitamin E significantly reduced the in vitro susceptibility of LDL to oxidation, which was not related to progression.In this trial results were stratified by genetic predisposition. Smokers lacking the detoxifying enzyme activity of glutathion S -transferase µ (genotype GSTM1- 0 ) were compared with those with the positive genotype (GSTM1- 1 ). In the GSTM1- 0 group vitamin E reduced the proportion of smokers with increased carotid IMT by 62% (p=0.06) for the left posterior and by 73% (p=0.01) for the left anterior wall. No effects were observed for the IMT at the right side.In a 3-month randomized double-blind placebo-controlled trial among 83 apparently healthy elderly, aged 67-87 years, 100 mg vitamin E supplementation significantly decreased the percentage of oxidized linoleic acid in LDL (10.4%) compared to the placebo group (4.6%). In this trial vitamin E supplementation did not affect the cellular and humoral immune response.In conclusion , no supportive evidence is provided that vitamin E supplementation has beneficial effects on atherosclerosis and immune response among smokers and elderly. The observations for GSTM1- 0 genotype needs further confirmation. The protective effect of vitamin E on LDL oxidation in vitro lacks validation in vivo. Research on biomarkers of lipid oxidation and arterial damage needs priority to more adequately assess the clinical relevance and optimal intake of vitamin E.</p

Multifaceted Nutritional Intervention In Hemodialysis Patients

Mortality rate in patients undergoing chronic hemodialysis (HD) in the United States remain unacceptably high despite improvement in dialysis technology. Cardiovascular disease (CVD) account for more than 50% of the premature death in this population. Evidences indicate that the accelerated CVD are attributed to by malnutrition, inflammation, dyslipidemia and oxidative stress. Dietary intervention using nutrients with antioxidant, anti-inflammatory, and potential lipid altering properties to correct the aforementioned problems remain inconclusive. Furthermore, such nutritional intervention trial is often hampered by poor compliance related to medical and socioeconomic barriers. Therefore, a series of randomized, double-blind, placebo-controlled, parallel trials were undertaken to document the technical feasibility of `directly observed treatment\u27 and the global impact of several nutrients namely omega-3 and vitamin E tocotrienols on nutritional and oxidative indicators, inflammatory markers and lipid profiles in a cohort of chronic HD patients. The objective of the first study was to evaluate the impact of omega-3 plus liquid protein supplement on serum albumin, plasma lipids and other indicator of nutrition and inflammation. markers. The study recruited 63 subjects and they were randomized into placebo+protein (n=32) and omega-3+protein (n=31) groups. The two intervention groups received 30mL of a liquid protein plus 2.4g omega-3 or placebo, three times per week after their routine dialysis session for 6 months. Directly observed nutritional supplement resulted in significant improvement in the LDLC/HDLC ratio in the omega-3 group as compared to the placebo group (P=0.043). In the omega-3 group, serum albumin was also marginally higher after 6 months as compared to the baseline (P=0.07). The observed increase in CRP levels in the placebo group over 6 months was not apparent in the omega-3 group, although there was no significant difference between groups. NFκB, MIS, nPNA, BMI and hemoglobin were unaffected by the intervention. Therefore, it is conclude that `directly observed treatment\u27 with an omega-3 based supplement (as opposed to a pure protein supplement) showed beneficial effects on lipid profile, and CRP levels. Further studies using a combination of outpatient and inpatient `directly observed treatment\u27 is warranted. Given a proven feasibility of directly observed treatment in the first study, we conducted a second study using tocotrienol rich fractions (TRF) by incorporating the same design to maximize compliance but with additional take home supplements. Vitamin E tocotrienols have been reported to confer anti-inflammatory, antioxidant and a potential of lipid altering benefits in vitro, in vivo and in some other clinical population. However, the impact of this nutrient in HD population is unknown. Subjects were provided daily with capsules containing either vitamin E tocotrienol-rich fraction (TRF) (180 mg tocotrienols, 40 mg tocopherols) or placebo (0.48 mg tocotrienols, 0.88 mg tocopherols). For the results, TRF supplementation did not impact any nutritional, inflammatory, or oxidative status biomarkers over time when compared with the baseline within the group (one-way repeated measures analysis of variance) or when compared with the placebo group at a particular time point (independent t-test). However, the TRF supplemented group showed improvement in lipid profiles after 12 and 16 weeks of intervention when compared with placebo at the respective time points. Normalized plasma triacylglycerols (cf baseline) in the TRF group was reduced by 33 mg/dL (P=0.032) and 36 mg/dL (P=0.072) after 12 and 16 weeks of intervention but no significant improvement was seen in the placebo group. Similarly, normalized plasma high-density lipoprotein cholesterol was higher (P\u3c0,05) in the TRF group as compared with placebo at both week 12 and week 16. The changes in the TRF group at week 12 and week 16 were associated with higher plasma apolipoprotein A1 concentration (P\u3c0.02) and lower cholesteryl-ester transfer protein activity (P\u3c0.001). As a conclusion, TRF supplementation improved lipid profiles in the study of maintenance HD patients. A multi-centered trial is warranted to confirm these observations. Finally, following a positive impact of TRF supplementation on lipid profiles, we undertook a metabolomics approach to investigate whether the TRF supplementation lead to overall changes in patients\u27 metabolomics profile and whether the observed changes in plasma lipids correlates with their metabolomics profile. Based on the principal component analysis (PCA), there was a separation pattern between the TRF and placebo groups at week-12. After applying partial least square-discriminant analysis (PLS-DA), there was a clear separation between the two groups indicating different metabolomics profiles. In addition, metabolomics profile in both TRF and placebo group was correlated with inflammatory markers and lipid profiles suggesting that some plasma metabolite could predict/ responsible for the changes in lipid profiles and inflammatory markers

Understanding the biochemical properties and physiological function of the protein syncollin

Syncollin is a 16-kDa protein that was originally isolated from the pancreatic zymogen granule. It is now known also to be expressed in the gut, the spleen and in neutrophils. The protein contains intramolecular disulphide bonds and is present both free within the ZG lumen and tightly associated with the luminal leaflet of the ZG membrane; it is also secreted into the pancreatic juice. Syncollin is able to oligomerize, and assemble into doughnut-shaped structures, which might explain its known pore-forming activity. Syncollin appears to be involved in a number of gut-based disease states. For instance, syncollin expression was found to be down-regulated in the colon when a bacterial suspension was administered to germ-free mice, and in mice with chemically-induced colitis-associated cancer. The available evidence suggests that syncollin plays an important role in the gut, and possibly elsewhere. This dissertation describes my attempts to understand this role and its structural basis. I first assessed various methods for purification of recombinant syncollin. Syncollin was expressed with various epitope tags (including His, GST and Strep) in bacteria, insect cells and mammalian cells. The best results were obtained by expressing syncollin bearing a double-Strep tag at its C terminus (syncollin-Strep) in mammalian (tsA-201) cells and purifying the protein from the cell supernatant using the Strep-Tactin XTTM system. Syncollin-Strep purified in this way contained intra-molecular disulphide bonds and recapitulated the ability of the native protein to bind to syntaxin 2 and permeabilize membranes. In the pancreatic juice, syncollin will encounter an environment rich in proteolytic activity. One might expect, therefore, that its structure would be highly stable. To test this hypothesis, I assessed the thermal stability of the protein using circular dichroism (CD) spectroscopy. The CD spectrum of syncollin-Strep indicated a predominantly beta-sheet structure. When the protein was subjected to a temperature ramp up to 90°C, the spectrum became flattened, although complete unfolding did not occur, indicating that the protein does indeed have a very high thermal stability. A model for syncollin, based on its primary sequence, predicts a predominantly beta-sheet structure, consistent with my CD data, and suggests the presence of intramolecular disulphide bonds. When I disrupted potential bonds by mutation of appropriate cysteine residues, syncollin-Strep retained its antibacterial efficacy, but its thermal stability was reduced, suggesting the involvement of disulphide bonding in stabilizing the structure of the protein. With regard to its potential role in the gut, I found that syncollin-Strep binds to bacterial peptidoglycan, and restricts the growth of representative Gram-positive (Lactococcus lactis) and Gram-negative (Escherichia coli) bacteria. Syncollin induces propidium iodide uptake into E. coli (but not L. lactis), indicating permeabilization of the bacterial membrane. In support of this idea, I confirmed that syncollin-Strep, like native syncollin, has pore-forming properties. Syncollin-Strep causes surface structural damage in both L. lactis and E. coli, as visualized by scanning electron microscopy. In addition, syncollin-Strep had additive effects on L. lactis when combined with either ampicillin (bactericidal) or tetracycline (bacteriostatic) in L. lactis. In light of these results, I propose that syncollin is a previously unidentified member of a large group of antimicrobial polypeptides that control the gut microbiome. I found that expression of syncollin in neutrophils is punctate and granular. Upon activation of the neutrophils, syncollin became mobilized at the plasma membrane, and was also secreted from the cells. Secreted syncollin bound to decondensed chromatin structures characteristic of neutrophil extracellular traps (NETs). Further, when neutrophils were activated in the presence of bacteria, the bacteria became coated with secreted syncollin, consistent with the anti-bacterial role proposed above. In conclusion, the results presented in this dissertation indicate that, through its antibacterial effects, syncollin plays a role in host defence in both the gut and the bloodstream

Advances in Lipidomics: Biomedicine, Nutrients and Methodology

This book contains 12 articles covering biomedicine, nutrition, and the methodology of lipidomics . These works were first published by MDPI in a Special Issue of Metabolites. Phospholipids, sphingolipids, glyosylinositolphosphoceramides, cholesteryl esters, acyl-carnitines, and oxylipins are within the lipid classes accounted for studies regarding liver disease, Wilson disease, kidney disease, cardiovascular disease, adipogenesis, and the role lipids play in cancer and virus infection. High-throughput lipid extraction and guidelines for lipid annotation are addressed in several papers. This book is expected to provide a comprehensive view of the diverse areas where lipidomics looms largest