Nm Siegart

Interleukin-6 in atherosclerosis: atherogenic or atheroprotective?

ABSTRACT Interleukin-6 (IL-6) is a unique pleiotropic cytokine exhibiting both pro- and anti-inflammatory properties depending on the target cell type. Plasma IL-6 levels are associated with cardiovascular risk. IL-6 elevation in atherosclerosis results in effects on multiple cells involved in lipid processing and plaque formation. IL-6 is also the primary determinant of acute phase protein production. IL-6 has a number of properties that foster development of cardiovascular disease. These include activation of endothelial cells, pro-thrombotic effects on platelets and promotion of smooth muscle proliferation and macrophage lipid accumulation. Despite these overall unfavourable effect on cells involved in atheroma formation, IL-6 also has a positive impact on the lipid handling system through upregulation of ATP binding cassette transporter (ABC)A1, a protein involved in macrophage lipid efflux. Further, IL-6 can inhibit other inflammatory cytokines. Based on its possible role in accelerating atherosclerosis, blockade of IL-6 action with the antibody tocalizumab, a treatment for rheumatoid arthritis and juvenile rheumatoid arthritis, has been evaluated as an atheroprotective agent, but studies are inconclusive. This review discusses multiple aspects of IL-6 effects on parameters related to development of atherosclerosis and highlights their manifestations in cell culture, murine models and human studies.

Resveratrol counters systemic lupus erythematosus-associated atherogenicity by normalizing cholesterol efflux.

Resveratrol is a bioactive molecule used in dietary supplements and herbal medicines and consumed worldwide. Numerous investigations by our group and others have indicated cardioprotective and anti-inflammatory properties of resveratrol. The present study explored potential atheroprotective actions of resveratrol on cholesterol efflux in cultured human macrophages exposed to plasma from systemic lupus erythematosus (SLE) patients. These results were confirmed in ApoE−/−Fas−/− double knockout mice, displaying a lupus profile with accelerated atherosclerosis. Resveratrol treatment attenuated atherosclerosis in these mice. THP-1 human macrophages were exposed to 10% pooled or individual plasma from patients who met diagnostic criteria for SLE. Expression of multiple proteins involved in reverse cholesterol transport (ABCA1, ABCG1, SR-B1, and cytochrome P450 27-hydroxylase) was assessed using QRT-PCR and Western blotting techniques. Ten-week-old ApoE−/−Fas−/− double knockout mice (n = 30) were randomly divided into two equal groups of 15, one of which received 0.01% resveratrol for 10 consecutive weeks. Atherosclerosis progression was evaluated in murine aortas. Bone marrow-derived macrophages (BMDM) were cultured and expression of cholesterol efflux proteins was analyzed in each group of mice. Our data indicate that inhibition of cholesterol efflux by lupus plasma in THP-1 human macrophages is rescued by resveratrol. Similarly, administration of resveratrol in a lupus-like murine model reduces plaque formation in vivo and augments cholesterol efflux in BMDM. This study presents evidence for a beneficial role of resveratrol in atherosclerosis in the specific setting of SLE. Therefore, resveratrol may merit investigation as an additional resource available to reduce lipid deposition and atherosclerosis in humans, especially in such vulnerable populations as lupus patients.

Amyloid toxicity in Alzheimer’s disease

Abstract A major feature of Alzheimer’s disease (AD) pathology is the plaque composed of aggregated amyloid-β (Aβ) peptide. Although these plaques may have harmful properties, there is much evidence to implicate soluble oligomeric Aβ as the primary noxious form. Aβ oligomers can be generated both extracellularly and intracellularly. Aβ is toxic to neurons in a myriad of ways. It can cause pore formation resulting in the leakage of ions, disruption of cellular calcium balance, and loss of membrane potential. It can promote apoptosis, cause synaptic loss, and disrupt the cytoskeleton. Current treatments for AD are limited and palliative. Much research and effort is being devoted to reducing Aβ production as an approach to slowing or preventing the development of AD. Aβ formation results from the amyloidogenic cleavage of human amyloid precursor protein (APP). Reconfiguring this process to disfavor amyloid generation might be possible through the reduction of APP or inhibition of enzymes that convert the precursor protein to amyloid.

BMI-DEPENDENT EFFECTS OF ADIPOSE TISSUE EXOSOMES ON HUMAN MACROPHAGE CHOLESTEROL TRANSPORT GENE EXPRESSION

Obesity, a major risk for atherosclerotic cardiovascular disease, has quadrupled in adolescents in the last 30 years. How adipose tissue influences the pathological process of atherosclerosis is not well understood. This study compares cholesterol efflux gene expression in human macrophages exposed

MP1: CORRECTING ATHEROGENIC EFFECTS OF LUPUS PLASMA ON MACROPHAGES WITH RESVERATROL AND MYCOPHENOLATE

Purpose of Study Premature atherosclerosis with coronary artery disease is a major cause of morbidity in Systemic Lupus Erythematosus (SLE). SLE patient plasma induces a pro-atherogenic profile of cholesterol transport genes in macrophages. A common immunosuppressive treatment for SLE, mycophenolate (MMF) reduces scavenger receptors thus reducing lipid influx. We have demonstrated atheroprotective properties of the polyphenol resveratrol on cholesterol efflux. This study determines whether MMF and resveratrol work synergistically to regulate cholesterol transport in macrophages exposed to pro-atherogenic SLE plasma. Methods Used THP-1 human macrophages (106/ml) were incubated in 10% SLE plasma with: media (control); MMF (1 µg/ml); resveratrol (50 µM); and MMF+resveratrol. After 24 h incubation, total RNA and protein were isolated. Message level of scavenger receptors CD36, LOX1, and SRA1; and efflux proteins 27-hydroxylase, ATP binding cassette transporter (ABC)A1, and ABCG1 were evaluated by QRT-PCR and confirmed by immunoblot. Cholesterol efflux was measured by Amplex Red Cholesterol Assay kit run±cholesterol esterase. Summary of Results In 10% SLE plasma, MMF suppressed efflux genes ABCA1 and ABCG1 (58.38±3.5% and 72.98±3.3%) vs. SLE plasma alone (p<0.0001) while MMF+resveratrol corrected this suppression. In SLE plasma, MMF+resveratrol decreased ScrA1 and LOX-1 by 15±2.5% and 47±1.0%, respectively vs. resveratrol alone (p<0.0001). SLE plasma promoted cholesterol accumulation in THP-1 macrophages and prevented efflux into medium. It increased the ratio of cholesterol esters to free cholesterol (ChE/FC). Resveratrol decreased intracellular cholesterol and restored ChE/FC ratios to that of cells in healthy control plasma. Conclusions MMF and resveratrol exhibit complimentary effects on macrophages exposed to SLE plasma. Both agents combined restore cholesterol influx and efflux gene expression to that of cells treated with control plasma. Resveratrol additionally reverses cholesterol accumulation caused by SLE plasma. Further evaluation of resveratrol+MMF in atherosclerosis in SLE may lead to improved treatment.

11: THE ADENOSINE A2A RECEPTOR AGONIST UK-432,097 STIMULATES ANTI-ATHEROGENIC REVERSE CHOLESTEROL TRANSPORT PROTEINS IN THP-1 HUMAN MACROPHAGES

Purpose of Study Methotrexate (MTX) is an anti-rheumatic drug with atheroprotective properties mediated through adenosine release and activation of the adenosine A2A receptor (A2AR). A2AR ligation increases reverse cholesterol transport via upregulation of cholesterol efflux proteins ATP-binding cassette transporter (ABC)A1 and ABCG1, liver X receptor (LXR) and cholesterol 27-hydroxylase. MTX is non-specific and associated with adverse effects on liver and kidney. Therefore, this study examines the anti-atherogenic efficacy of a specific A2AR agonist, UK-432,097, a drug with an established safety profile in humans. Methods Used THP-1 human macrophages were incubated in the following conditions: (1) RPMI media (untreated control); (2) dimethyl sulfoxide vehicle control; (3) UK-432,097 (100 nM); (4) ZM-241385 (1 µM) (A2AR antagonist)+UK-432,097 (100 nM). Gene expression analysis was performed using QRT-PCR for cholesterol efflux genes, normalized to the housekeeping gene GAPDH. Western blotting was performed using specific antibodies. All data were analyzed by one-way ANOVA with P values <0.05 considered significant. Summary of Results Following 6 h exposure to UK-432,097, mRNA and protein levels of ABCA1 increased by 88.75±5.4% and by 56.34±12.4% above control (P<0.01), respectively. ABCG1 expression increased by 58.42±6.32% and 65.45±5.24% vs. control (P<0.01), respectively. Following 18 h incubation in UK-432,097, 27-hydroxylase mRNA and protein increased by 46.45±3.4% and 50.27±8.9% (P<0.01), respectively. Message and protein level of LXRα were upregulated to 155.80±4.9% and 157.98±12.9% (n=3, P<0.01), respectively. A2AR blockade with ZM-241385 negated the effect of UK-432,097. UK-432,097 decreased oxidized LDL uptake by 28.9% in THP-1 macrophages (P<0.01). Conclusions This study demonstrates that UK-432,097 increases anti-atherogenic reverse cholesterol transport proteins with concomitant reduction in oxidized lipid accumulation in THP-1 macrophages. Since MTX is already being used in clinical trials to reduce cardiovascular risk, our results encourage further studies of specific A2AR agonists as cardioprotective treatment in high risk individuals.

1: COMPARATIVE EFFECT OF SELECTIVE AND NON-SELECTIVE COX-2 INHIBITORS ON LIPID ACCUMULATION IN HUMAN MACROPHAGES

Purpose of Study It is the second decade of controversy regarding the cardiovascular (CV) effects of cycloxygenase-2 (COX-2) inhibitors. COX-2 inhibitors possess anti-inflammatory and analgesic effects comparable with conventional non-steroidal anti-inflammatory drugs, but produce fewer gastrointestinal adverse effects. Here we demonstrate that only selective COX-2 inhibitors cause disruption of the delicate balance between cholesterol efflux and influx that leads to lipid overload and macrophage foam cell formation (FCF). Methods Used THP-1 human macrophages were incubated with: celecoxib (10 µM, 25 µM); rofecoxib (10 µM, 25 µM); naproxen (10 µM, 25 µM); acetaminophen (0.5 mM, 1 mM)±oxidized low density lipoprotein (oxLDL, 25 µg/ml, 48 h) or 5 µg/ml (Dil)-oxLDL. FCF (% oil red O stained cells) and oxLDL accumulation were determined (fluorescent intensity). Scavenger receptors: CD36, LOX-1, SR-A1 and CXCL16 and cholesterol efflux proteins: ATP-binding cassette transporter (ABC) A1 and ABCG1 were detected in macrophages by QRT-PCR and immunocytochemistry. Summary of Results Celecoxib decreased ABCA1 and ABCG1 message in a concentration dependent manner: 68.2±13.36% for ABCA1 and 65.7±13.36% for ABCG1 (control set at 100%, n=6, P<0.01). Neither naproxen nor acetaminophen significantly affected expression of cholesterol efflux proteins. Both specific and nonspecific COX-2 inhibitors had a significant impact on expression of scavenger receptors CD36, LOX-1 and SR-A1–nearly double control (n=6, P<0.05). However, only specific COX-2 inhibitors significantly increased FCF in THP-1 differentiated macrophages (62.2±5.2% for celecoxib and 56.3±3.4% for rofecoxib vs. 33.5±5.1% for untreated cells, P<0.05). Conclusions Here we report that only specific COX-2 inhibitors might contribute to atherogenesis by promoting lipid overload and lipoprotein accumulation. This may explain, in part, the increased CV risk in patients taking COX-2 inhibitors for extended periods. Despite increased scavenger receptor expression, naproxen and acetaminophen do not impact lipid content, perhaps because efflux pathways remain intact.

3: INHIBITION OF THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS RESTORES CHOLESTEROL EFFLUX IN THP-1 HUMAN MACROPHAGES EXPOSED TO PLASMA FROM TYPE 1 DIABETES MELLITUS PATIENTS

Purpose of Study Advanced glycation end products (AGE), proteins formed by nonenzymatic glycation, are prevalent in patients with diabetes mellitus (DM) and contribute to the development of atherosclerosis. We have demonstrated that plasma from patients with type 1 DM (T1DM) decreases expression of cholesterol efflux proteins in cultured THP-1 macrophages compared to healthy control (HC) plasma. Here we explore a mechanism that restores cholesterol efflux in T1DM plasma through blockade of the receptor for AGE (RAGE). Methods Used Carboxy-methyl-lysine-modified proteins (CML-MP), the most prevalent AGE in vivo, were measured in the plasma of 20 pediatric T1DM patients and 20 sex and age-matched HC by ELISA. THP-1 macrophages (106/ml) were incubated for 18 h in RPMI media in the presence of 10% plasma from each enrolled patient in triplicate±anti-RAGE antibody. Cholesterol efflux proteins: ATP binding cassette transporter (ABC)A1 and 27-hydroxylase were quantified by real-time RT-PCR using specific primers for each gene. Summary of Results The level of CML-MP was significantly elevated in T1DM plasma (1.65±1.3 ng/ml) versus HC plasma (1.05±0.4 ng/ml) (P<0.05, n=20). ABCA1 expression was significantly lower in macrophages exposed to T1DM plasma (1.108±0.8 U) versus HC plasma (1.624±0.6) (P<0.05, n=20). Exposure of THP-1 macrophages to T1DM plasma downregulated 27-hydroxylase mRNA to 1.94±0.9 U in T1DM versus 3.4±2.9 U in HC (P<0.01, n=20). Inactivation of RAGE before exposure to T1DM plasma increased ABCA1 expression by 20%. However, we observed no effect on the level of 27-hydroxylase. Conclusions We demonstrate that elevated AGE in plasma of T1DM patients inhibits cholesterol efflux and suppresses intracellular cholesterol processing via 27-hydroxylase and ABCA1 in naïve macrophages. RAGE inactivation restores mRNA level of the ABCA1 transporter, but not the 27-hydroxylase enzyme. These findings impart new targets for prevention of cardiovascular disease in DM and suggest that factors other than AGE may impact cholesterol transport.