Chandrakala Aluganti Narasimhulu

Oxidatively Modified Low Density Lipoprotein (LDL) Inhibits TLR2 and TLR4 Cytokine Responses in Human Monocytes but Not in Macrophages

Background: The role of oxidatively modified LDL (oxmLDL) in atherosclerotic inflammation is controversial. Results: Endotoxin-free oxmLDL fails to enhance mononuclear phagocyte inflammatory responses but suppresses IL-1β, TNF, and IL-6 when combined with endotoxin or PamCSK4. Conclusion: OxmLDL suppresses proinflammatory responses. Significance: Although LDL preparations have been linked to inflammatory responses, endotoxin-free, oxidatively modified LDL inhibits monocyte Toll ligands. Inflammation characterized by the expression and release of cytokines and chemokines is implicated in the development and progression of atherosclerosis. Oxidatively modified low density lipoproteins, central to the formation of atherosclerotic plaques, have been reported to signal through Toll-like receptors (TLRs), TLR4 and TLR2, in concert with scavenger receptors to regulate the inflammatory microenvironment in atherosclerosis. This study evaluates the role of low density lipoproteins (LDL) and oxidatively modified LDL (oxmLDL) in the expression and release of proinflammatory mediators IκBζ, IL-6, IL-1β, TNFα, and IL-8 in human monocytes and macrophages. Although standard LDL preparations induced IκBζ along with IL-6 and IL-8 production, this inflammatory effect was eliminated when LDL was isolated under endotoxin-restricted conditions. However, when added with TLR4 and TLR2 ligands, this low endotoxin preparation of oxmLDL suppressed the expression and release of IL-1β, IL-6, and TNFα but surprisingly spared IL-8 production. The suppressive effect of oxmLDL was specific to monocytes as it did not inhibit LPS-induced proinflammatory cytokines in human macrophages. Thus, TLR ligand contamination of LDL/oxmLDL preparations can complicate interpretations of inflammatory responses to these modified lipoproteins. In contrast to providing a proinflammatory function, oxmLDL suppresses the expression and release of selected proinflammatory mediators.

Anti-atherosclerotic and anti-inflammatory actions of sesame oil.

Atherosclerosis, a major form of cardiovascular disease, has now been recognized as a chronic inflammatory disease. Nonpharmacological means of treating chronic diseases have gained attention recently. We previously reported that sesame oil has anti-atherosclerotic properties. In this study, we have determined the mechanisms by which sesame oil might modulate atherosclerosis by identifying genes and inflammatory markers. Low-density lipoprotein receptor knockout (LDLR(-/-)) female mice were fed with either an atherogenic diet or an atherogenic diet reformulated with sesame oil (sesame oil diet). Plasma lipids and atherosclerotic lesions were quantified after 3 months of feeding. Plasma samples were used for cytokine analysis. RNA was extracted from the liver tissue and used for global gene arrays. The sesame oil diet significantly reduced atherosclerotic lesions, plasma cholesterol, triglyceride, and LDL cholesterol levels in LDLR(-/-) mice. Plasma inflammatory cytokines, such as MCP-1, RANTES, IL-1α, IL-6, and CXCL-16, were significantly reduced, demonstrating an anti-inflammatory property of sesame oil. Gene array analysis showed that sesame oil induced many genes, including ABCA1, ABCA2, APOE, LCAT, and CYP7A1, which are involved in cholesterol metabolism and reverse cholesterol transport. In conclusion, our studies suggest that a sesame oil-enriched diet could be an effective nonpharmacological treatment for atherosclerosis by controlling inflammation and regulating lipid metabolism.

Aspirin may promote mitochondrial biogenesis via the production of hydrogen peroxide and the induction of Sirtuin1/PGC-1α genes

Based on the rapid hydrolysis of acetyl salicylic acid (ASA, Aspirin) to salicylic acid (SA), the ability of SA to form dihydroxy benzoic acid (DBA), and the latters redox reactions to yield hydrogen peroxide (H(2)O(2)), we predicted that ASA may have the potential to induce Sirtuin1 (Sirt1) and its downstream effects. We observed that treatment of cultured liver cells with ASA resulted in the induction of Sirt1, peroxisome proliferator-activated receptor-gamma co-activator-1α (PGC-1α), and NAD(P)H quinone oxidoreductase 1 (Nqo1) genes. Paraoxonase 1 (PON1) and Aryl hydrocarbon receptor (AhR) siRNA transfections inhibited the induction of gene expressions by ASA suggesting the need for the acetyl ester hydrolysis and hydroxylation to DHBA. The latter also induced Sirt1, confirming the proposed pathway. As predicted, ASA and SA treatment resulted in the production of H(2)O(2), a known inducer of Sirt1 and confirmed in the current studies. More importantly, ASA treatment resulted in an increase in mitochondria as seen by tracking dyes. We suggest that DHBA, generated from ASA, via its oxidation/reduction reactions mediated by Nqo1 might be involved in the production of O(2)(-.) and H(2)O(2). As Sirt1 and PGC-1α profoundly affect mitochondrial metabolism and energy utilization, ASA may have therapeutic potential beyond its ability to inhibit cyclooxygenases.

Cell membrane damage is involved in the impaired survival of bone marrow stem cells by oxidized low‐density lipoprotein

Cell therapy with bone marrow stem cells (BMSCs) remains a viable option for tissue repair and regeneration. A major challenge for cell therapy is the limited cell survival after implantation. This study was to investigate the effect of oxidized low‐density lipoprotein (ox‐LDL, naturally present in human blood) on BMSC injury and the effect of MG53, a tissue repair protein, for the improvement of stem cell survival. Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox‐LDL, which caused significant cell death as reflected by the increased LDH release to the media. Exposure of MAPCs to ox‐LDL led to entry of fluorescent dye FM1‐43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox‐LDL also generated reactive oxygen species (ROS) as measured with electron paramagnetic resonance spectroscopy. While antioxidant N‐acetylcysteine completely blocked ROS production from ox‐LDL, it failed to prevent ox‐LDL‐induced cell death. When MAPCs were treated with the recombinant human MG53 protein (rhMG53) ox‐LDL induced LDH release and FM1‐43 dye entry were significantly reduced. In the presence of rhMG53, the MAPCs showed enhanced cell survival and proliferation. Our data suggest that membrane damage induced by ox‐LDL contributed to the impaired survival of MAPCs. rhMG53 treatment protected MAPCs against membrane damage and enhanced their survival which might represent a novel means for improving efficacy for stem cell‐based therapy for treatment of diseases, especially in setting of hyperlipidemia.

Atherosclerosis--do we know enough already to prevent it?

In this review, we have briefly summarized the characteristics of lipids and lipoproteins and the atherosclerotic process. The development of atherosclerosis is a continuous process that involves numerous cellular and acellular processes that influence the behavior of each other. These include oxidative stress, lipoprotein modifications, macrophage polarization, macrophage lipid accumulation, generation of pro- and anti-inflammatory components, calcification, cellular growth and proliferation, and plaque rupture. The precise role(s) of many of these are unknown. Understanding the events at each particular stage might shed more light onto the process as a whole and could potentially reveal targets for intervention. Therapeutic modalities that work at one stage may have little to no influence on other stages of the disease.

Novel technique for generating macrophage foam cells for in vitro reverse cholesterol transport studies

Generation of foam cells, an essential step for reverse cholesterol transport studies, uses the technique of receptor-dependent macrophage loading with radiolabeled acetylated LDL. In this study, we used the ability of a biologically relevant detergent molecule, lysophosphatidylcholine (lyso-PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabeled cholesterol/lyso-PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month of storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled, or radiolabeled)/lyso-PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by TLC. Such foam cells unloaded cholesterol when incubated with HDL but not with oxidized HDL. We propose that stable cholesterol or CE/lyso-PtdCho micelles would offer advantages over existing methods.

Differential lipid metabolism in monocytes and macrophages: influence of cholesterol loading

The influence of the hypercholesterolemia associated with atherosclerosis on monocytes is poorly understood. Monocytes are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PC). Indeed, in line with recent reports, we found that monocytes accumulate cholesteryl esters (CEs) in hypercholesterolemic mice, demonstrating the need for studies that analyze the effects of lipid accumulation on monocytes. Here we analyze the effects of cholesterol and lyso-PC loading in human monocytes and macrophages. We found that cholesterol acyltransferase and CE hydrolase activities are lower in monocytes. Monocytes also showed a different expression profile of cholesterol influx and efflux genes in response to lipid loading and a different pattern of lyso-PC metabolism. In monocytes, increased levels of CE slowed the conversion of lyso-PC into PC. Interestingly, although macrophages accumulated glycerophosphocholine, phosphocholine was the main water-soluble choline metabolite being generated in monocytes, suggesting a role for mono- and diacylglycerol in the chemoattractability of these cells. In summary, monocytes and macrophages show significant differences in lipid metabolism and gene expression profiles in response to lipid loading. These findings provide new insights into the mechanisms of atherosclerosis and suggest potentials for targeting monocyte chemotactic properties not only in atherosclerosis but also in other diseases.

Probucol Protects Endothelial Progenitor Cells Against Oxidized Low-Density Lipoprotein via Suppression of Reactive Oxygen Species Formation In Vivo.

Background/Aims: Oxidized low-density lipoprotein (ox-LDL) is a major component of hyperlipidemia and contributes to atherosclerosis. Endothelial progenitor cells (EPCs) play an important role in preventing atherosclerosis and notably decreased in hyperlipidemia. Ox-LDL and ox-LDL-related reactive oxygen species (ROS) have deleterious effects on EPCs. Probucol as an antioxidant and anti-inflammatory drug reduces ROS production. The present study was to determine if probucol could protect EPCs from ox-LDL in vivo and to investigate the potential mechanisms. Methods: ox-LDL was injected into male C57BL/6 mice for 3 days with or without probucol treatment with PBS as control. Bone marrow (BM) fluid, serum, circulating mononuclear cells (MNCs) and EPCs were collected for analysis. Results: the increased extracellular ROS in BM, serum and blood intracellular ROS production in the mice with ox-LDL treatment in association with a significant reduction of circulating MNCs and EPCs were restored with Probucol treatment. A significant increase in the serum ox-LDL and C-reactive protein and decrease in superoxide dismutase and circulating MNCs and EPCs were observed in hyperlipidemic patients that were effectively reversed with probucol treatment. Conclusion: these data suggested that probucol could protect EPCs from ox-LDL through inhibition of ROS production in vivo.

Aspirin may influence cellular energy status

In our previous findings, we have demonstrated that aspirin/acetyl salicylic acid (ASA) might induce sirtuins via aryl hydrocarbon receptor (Ah receptor). Induction effects included an increase in cellular paraoxonase 1 (PON1) activity and apolipoprotein A1 (ApoA1) gene expression. As predicted, ASA and salicylic acid (SA) treatment resulted in generation of H2O2, which is known to be an inducer of mitochondrial gene Sirt4 and other downstream target genes of Sirt1. Our current mass spectroscopic studies further confirm the metabolism of the drugs ASA and SA. Our studies show that HepG2 cells readily converted ASA to SA, which was then metabolized to 2,3-DHBA. HepG2 cells transfected with aryl hydrocarbon receptor siRNA upon treatment with SA showed the absence of a DHBA peak as measured by LC-MS/MS. MS studies for Sirt1 action also showed a peak at 180.9 m/z for the deacetylated and chlorinated product formed from N-acetyl lε-lysine. Thus an increase in Sirt4, Nrf2, Tfam, UCP1, eNOS, HO1 and STAT3 genes could profoundly affect mitochondrial function, cholesterol homeostasis, and fatty acid oxidation, suggesting that ASA could be beneficial beyond simply its ability to inhibit cyclooxygenase.

Increased presence of oxidized low‐density lipoprotein in the left ventricular blood of subjects with cardiovascular disease

Oxidized LDL (Ox‐LDL) and oxidative stress have been implicated in both atherosclerosis and congestive heart failure (HF) development. Here, we tested whether Ox‐LDL levels in left ventricular blood (LVB) might differ from those of venous peripheral blood (PB), and whether the level might depend on cardiac function. We also tested whether the LDL molecule is likely to have a longer residence time in the left ventricle of HF subjects with low ejection fraction (EF). The aim of this study was to determine Ox‐LDL levels, paraoxonase 1 (PON1) activity, and cholesterol efflux capacity (CEC) of PB and LVB, and correlate these values with LVEF. Sixty‐one HF patients underwent preoperative transthoracic echocardiographic assessment of ventricular function. LVEFs were determined using Simpsons biplane technique. LVB and PB levels of Ox‐LDL were determined, and PON1 activity and plasma cholesterol efflux capacity were measured. A significant increase in the levels of Ox‐LDL in LVB was noted as compared to levels in PB, even when EF was near normal. However, as ejection fraction decreased, the level of Ox‐LDL in PB approached that of the LVB. PON1 activity and cholesterol efflux studies indicated increased oxidative stress in LVB and a decreased ability to promote cholesterol efflux from lipid‐enriched macrophages. The results suggest that LVB is more oxidatively stressed compared to PB, and therefore LV tissue might be affected differently than peripheral tissues. We recently reported that brain natriuretic peptide (BNP), a marker for HF, is induced by Ox‐LDL, so it is possible localized factors within the LV could profoundly affect markers of HF.