Intracellular generation of MDA-lys epitope in foam cells

Huang, Guo-Fang; Wang, Zwei-Ping M.; Wang, She-Chuan; Chu, Redman; Mao, Simon J.T.

doi:10.1016/s0024-3205(99)00247-7

Cited by 10 publications

(8 citation statements)

References 38 publications

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“…Our results demonstrate that the internalization of cholesterol – without oxidative modification – by macrophages is sufficient to trigger cellular reactions leading to the generation of superoxide. We have reported previously that, upon internalization, ac‐LDL generates the MDA‐lysine epitope in foam cells [41]. It is plausible to suggest that cholesterol loading induces superoxide production, lipid peroxidation, and subsequent generation of MDA‐modification to proteins.…”

Section: Resultsmentioning

confidence: 93%

“…The non‐adherent cells were removed and the monolayers were then placed in DMEM containing 10% fetal bovine serum and supplemented with 100 μg/ml ox‐LDL or ac‐LDL. Foam cell formation was monitored by Oil Red O staining [41]. Monolayers of macrophages on glass cover slips were fixed with 10% formaldehyde in PBS (pH 7.4) for 10 min at room temperature, stained with Oil Red O, counterstained for 10 min with hematoxylin, and then examined under a microscope.…”

Section: Methodsmentioning

confidence: 99%

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Cholesterol loading augments oxidative stress in macrophages

2006

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To investigate the molecular consequence of loading free cholesterol into macrophages, we conducted a large-scale gene expression study to analyze acetylated-LDL-laden foam cells (AFC) and oxidized-LDL-laden foam cells (OFC) induced from human THP-1 cell lines. Cluster analysis was performed using 9600-gene microarray datasets from time course experiment. AFC and OFC shared common expression profiles; however, there were sufficient differences between these two treatments that AFC and OFC appealed as two separate entities. We identified 80 commonly upregulated genes and 48 commonly downregulated genes in AFC and OFC. Functional annotation of the differentially expressed genes indicated that apoptosis, extracellular matrix, oxidative stress, and cell proliferation was deregulated. We also identified 87 differentially expressed genes unique for AFC and 31 genes for OFC. The uniquely expressed genes of AFC are associated with kinase activity, ATP binding activity, and transporter activity, while unique genes for OFC are associated with cell signaling and adhesion. To validate the hypothesis that oxidative stress is a common feature for AFC and OFC, we performed a cluster analysis employing the genes related to oxidative stress, but we were unable to distinguish AFC from OFC in this manner. We performed real-time RT-PCR and ELISA on foam cells to examine the transcripts and secreted protein of interleukin 1 beta (IL1b). IL1b was rapidly induced in foam cells, but for AFC both RNA level and protein level dropped immediately and was attenuated. To detect levels of reactive oxygen species in foam cells we conducted hydroethidine staining and observed high levels of superoxide anion. We conclude that loading free cholesterol induces high levels of superoxide anion, increases oxidative stress, and triggers a transient inflammatory response in macrophages.

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Section: Resultsmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Cholesterol loading augments oxidative stress in macrophages

2006

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“…Seven days following the final booster, blood was collected in 0.1% EDTA and plasma was obtained. The titer of this antibody was over 1:20,000 as judged by an ELISA using a method previously described (Huang et al, 1999).…”

Section: Preparation Of Antiserum Against Lgmentioning

confidence: 99%

β-Lactoglobulin is a Thermal Marker in Processed Milk as Studied by Electrophoresis and Circular Dichroic Spectra

Chen

Hwang

Liau

et al. 2005

Journal of Dairy Science

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As much of the sterilization process involves heat treatment during the preparation of milk on an industrial scale, the unpredictable measures of the process are an essential issue in determining the quality of the milk. The purpose of the present study was to investigate the major protein change(s) of whey proteins in processed milk and extend the knowledge for future reference in the dairy industry. Using a native polyacrylamide gel electrophoresis, we showed almost a 90% loss and denaturation of beta-lactoglobulin (LG), but not alpha-lactalbumin (LA), in some brands of the processed and dry milks. Immunochemical analysis using Western blotting revealed that part of the loss was attributed to the formation of large multiple forms of LG in the processed product. Such denaturation was presumably associated with the heating procedure used in the process. Essentially, LG was the only major fraction converted to aggregates in milk heated at 95 degrees C for 30 min on 2-dimensional PAGE. The detailed thermal denaturation of purified LG and LA at various temperatures (50 to 95 degrees C) and time (5 to 960 s) were investigated using a circular dichroic analysis. The maximal changes of ellipticity at 205 nm (converting beta-structure to disordered structure) were correlated to heating temperature and time. There were no significant conformational changes of LG at temperatures below 70 degrees C for as long as 480 s. Pronounced and rapid changes occurred between 80 to 95 degrees C in a time-dependent manner. Fifty percent of the maximal changes could be reached within 15 s. In conclusion, the unique chemical and immunochemical loss and conformational changes made LG a superior marker for evaluating the thermal processing of milk. The detailed thermal denaturation curves of LG constructed with its time and temperature in this study provide a valuable reference for the dairy industry. We postulate that heat treatment over 80 degrees C in 15 s may induce a significant denaturation of milk LG.

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“…The biological functions of MDA-Lys [N ε -(2-propenal) lysine] adducts generated with proteins have not been well studied. MDA-Lys epitopes are, however, closely associated with atherogenesis (1,2,4) and recognized as markers of oxidation. Oxidative stress is implicated in the pathogenesis of numerous diseases, including diabetes and atherosclerosis.…”

mentioning

confidence: 99%

“…They form Schiff-base adducts with Lys residues and cross-links with proteins in vitro and in vivo. HNE also reacts with Lys residues, primarily via Michael addition reaction (1)(2)(3)(4). These lipid-protein adducts are termed ALEs.…”

mentioning

confidence: 99%

Proinflammatory Effects of Advanced Lipoxidation End Products in Monocytes

Shanmugam

Figarola

et al. 2008

Diabetes

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OBJECTIVE—The reactions of carbohydrate- or lipid-derived intermediates with proteins lead to the formation of Maillard reaction products, which subsequently leads to the formation of advanced glycation/lipoxidation end products (AGE/ALEs). Levels of AGE/ALEs are increased in diseases like diabetes. Unlike AGEs, very little is known about ALE effects in vitro. We hypothesized that ALEs can have proinflammatory effects in monocytes. RESEARCH DESIGN AND METHODS—In a profiling approach, conditioned media from THP-1 cells either cultured in normal glucose (5.5 mmol/l) or treated with MDA-Lys or MDA alone were hybridized to arrays containing antibodies to 120 known human cytokines/chemokines. Pathway analyses with bioinformatics software were used to identify signalling networks. RESULTS—Synthetic ALE (malondialdehyde-lysine [MDA-Lys]) (50 μmol/l) could induce oxidant stress and also activate the transcriptional factor nuclear factor-κB (NF-κB) in THP-1 monocytes. MDA-Lys also significantly increased the expression of key candidate proinflammatory genes, interferon-γ–inducible protein-10, β1- and β2-integrins, cyclooxygenase-2 (COX-2), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 and -8, and inducible nitric-oxide synthase, which are also associated with monocyte dysfunction. Several key target proinflammatory proteins were significantly induced by MDA-Lys relative to normal glucose or MDA alone, including MCP-1; tumor necrosis factor ligand superfamily member-14; chemokine CC motif ligand-11 (CCL11); growth-related oncogene-α, -β, and -γ; and chemokine CXC motif ligand-13. Bioinformatics analyses identified a network of chemokine signaling among MDA-Lys–regulated genes. MDA-Lys also increased monocyte binding to vascular smooth muscle and endothelial cells. Furthermore, plasma from diabetic rats showed significantly higher levels of MDA-Lys and CCL11. CONCLUSIONS—These new results suggest that ALEs can promote monocyte activation and vascular complications via induction of inflammatory pathways and networks.

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Intracellular generation of MDA-lys epitope in foam cells

Cited by 10 publications

References 38 publications

Cholesterol loading augments oxidative stress in macrophages

Cholesterol loading augments oxidative stress in macrophages

β-Lactoglobulin is a Thermal Marker in Processed Milk as Studied by Electrophoresis and Circular Dichroic Spectra

Proinflammatory Effects of Advanced Lipoxidation End Products in Monocytes

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