Allison B. Reiss

Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells.

Adenosine, acting at its receptors, particularly A2A receptors, is a potent endogenous anti-inflammatory agent that modulates the functions and differentiation of inflammatory and immune cells. Because the inflammatory milieu abounds in proinflammatory cytokines, we investigated the effects of Th1-inflammatory cytokines on function and expression of adenosine A2A receptors in the human monocytic cell line THP-1. We found that, consistent with previous reports, adenosine and 2-[p-(2-carnonylethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680), a selective A2A receptor agonist, suppress IL-12 production but increase IL-10 production in LPS-activated THP-1 cells. These effects were blocked by the A2A receptor antagonist 4-{2-[7-amino-2-(2-furyl)[1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol (ZM-241385). More importantly, the suppressive effect of adenosine and CGS-21680 on IL-12 production was significantly enhanced in cells pretreated with either IL-1 (10 U/ml) or TNF-α (100 U/ml) but markedly attenuated in cells pretreated with IFN-γ (100 U/ml). Similarly, IL-1 and TNF-α treatment potentiated the stimulatory effect of adenosine and CGS-21680 on IL-10 production, whereas IFN-γ treatment almost completely abolished this effect. CGS-21680 stimulated an increase in intracellular cAMP in a time- and dose-dependent manner in IL-1- and TNF-α-treated cells but not in control or IFN-γ-treated cells. Both IL-1 and TNF-α increased A2A receptor mRNA and protein. In parallel with its effect on A2A receptor function, IFN-γ down-regulated A2A receptor message and protein. Because adenosine mediates many of the antiinflammatory effects of drugs such as methotrexate, these observations suggest that local changes in the cytokine milieu may influence the therapeutic response to those drugs by altering the expression and function of adenosine receptors on inflammatory cells.

Adenosine A2A receptors play a role in the pathogenesis of hepatic cirrhosis

1 Adenosine is a potent endogenous regulator of inflammation and tissue repair. Adenosine, which is released from injured and hypoxic tissue or in response to toxins and medications, may induce pulmonary fibrosis in mice, presumably via interaction with a specific adenosine receptor. We therefore determined whether adenosine and its receptors contribute to the pathogenesis of hepatic fibrosis. 2 As in other tissues and cell types, adenosine is released in vitro in response to the fibrogenic stimuli ethanol (40 mg dl−1) and methotrexate (100 nM). 3 Adenosine A2A receptors are expressed on rat and human hepatic stellate cell lines and adenosine A2A receptor occupancy promotes collagen production by these cells. Liver sections from mice treated with the hepatotoxins carbon tetrachloride (CCl4) (0.05 ml in oil, 50 : 50 v : v, subcutaneously) and thioacetamide (100 mg kg−1 in PBS, intraperitoneally) released more adenosine than those from untreated mice when cultured ex vivo. 4 Adenosine A2A receptor‐deficient, but not wild‐type or A3 receptor‐deficient, mice are protected from development of hepatic fibrosis following CCl4 or thioacetamide exposure. 5 Similarly, caffeine (50 mg kg−1 day−1, po), a nonselective adenosine receptor antagonist, and ZM241385 (25 mg kg−1 bid), a more selective antagonist of the adenosine A2A receptor, diminished hepatic fibrosis in wild‐type mice exposed to either CCl4 or thioacetamide. 6 These results demonstrate that hepatic adenosine A2A receptors play an active role in the pathogenesis of hepatic fibrosis, and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis.

Atheroprotective effects of methotrexate on reverse cholesterol transport proteins and foam cell transformation in human THP-1 monocyte/macrophages.

OBJECTIVE To determine whether methotrexate (MTX) can overcome the atherogenic effects of cyclooxygenase 2 (COX-2) inhibitors and interferon-gamma (IFNgamma), both of which suppress cholesterol efflux protein and promote foam cell transformation in human THP-1 monocyte/macrophages. METHODS Message and protein levels of the reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFNgamma, with and without MTX. Foam cell transformation of lipid-laden THP-1 macrophages was detected with oil red O staining and light microscopy. RESULTS MTX increased 27-hydroxylase message and completely blocked NS398-induced down-regulation of 27-hydroxylase (mean +/- SEM 112.8 +/- 13.1% for NS398 plus MTX versus 71.1 +/- 4.3% for NS398 alone; P < 0.01). MTX also negated COX-2 inhibitor-mediated down-regulation of ABCA1. The ability of MTX to reverse inhibitory effects on 27-hydroxylase and ABCA1 was blocked by the adenosine A2A receptor-specific antagonist ZM241385. MTX also prevented NS398 and IFNgamma from increasing transformation of lipid-laden THP-1 macrophages into foam cells. CONCLUSION This study provides evidence supporting the notion of an atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes reverse cholesterol transport and limits foam cell formation in THP-1 macrophages. This is the first reported evidence that any commonly used medication can increase expression of antiatherogenic reverse cholesterol transport proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular disease in rheumatoid arthritis patients is through facilitation of cholesterol outflow from cells of the artery wall.

Cholesterol in neurologic disorders of the elderly: stroke and Alzheimer’s disease

Mechanisms for the regulation of intracellular cholesterol levels in various types of brain and vascular cells are of considerable importance in our understanding of the pathogenesis of a variety of diseases, particularly atherosclerosis and Alzheimers disease (AD). It is increasingly clear that conversion of brain cholesterol into 24-hydroxycholesterol and its subsequent release into the periphery is important for the maintenance of brain cholesterol homeostasis. Recent studies have shown elevated plasma concentrations of 24-hydroxycholesterol in patients with AD and vascular dementia, suggesting increased brain cholesterol turnover during neurodegeneration. The oxygenases involved in the degradation and excretion of cholesterol, including the cholesterol 24-hydroxylase and the 27-hydroxylase, are enzymes of the cytochrome P-450 family. This review focuses on the newly recognized importance of cholesterol and its oxygenated metabolites in the pathogenesis of ischemic stroke and AD. The reduction in stroke and AD risk in patients treated with cholesterol-lowering statins is also discussed.

Adenosine A2A receptor occupancy stimulates expression of proteins involved in reverse cholesterol transport and inhibits foam cell formation in macrophages

Transport of cholesterol out of macrophages is critical for prevention of foam cell formation, the first step in the pathogenesis of atherosclerosis. Proteins involved in this process include cholesterol 27‐hydroxylase and adenosine 5′‐triphosphat‐binding cassette transporter A1 (ABCA1). Proinflammatory cytokines and immune complexes (IC) down‐regulate cholesterol 27‐hydroxylase and impede cholesterol efflux from macrophages, leading to foam cell formation. Prior studies have suggested occupancy of the anti‐inflammatory adenosine A2A receptor (A2AR) minimizes early atherosclerotic changes in arteries following injury. We therefore asked whether A2AR occupancy affects macrophage foam cell formation in response to IC and the cytokine interferon‐γ. We found that the selective A2AR agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamido‐adenosine (CGS‐21680) inhibited foam cell formation in stimulated THP‐1 human macrophages, and the effects of CGS‐21680 were reversed by the selective A2AR antagonist 4‐(2‐[7‐amino‐2‐(2‐furyl) [1, 2, 4]triazolo[2,3‐a] [1, 3, 5]triazin‐5‐ylamino]ethyl)phenol. In confirmation of the role of A2AR in prevention of foam cell formation, CGS‐21680 also inhibited foam cell formation in cultured murine peritoneal macrophages but did not affect foam cell formation in A2AR‐deficient mice. Agents that increase foam cell formation also down‐regulate cholesterol 27‐hydroxylase and ABCA1 expression. Therefore, we determined the effect of A2AR occupancy on expression of these reverse cholesterol transport (RCT) proteins and found that A2AR occupancy stimulates expression of message for both proteins. These results indicate that one mechanism for the antiatherogenic effects of adenosine is stimulation of the expression of proteins involved in RCT. These findings suggest a novel approach to the development of agents that prevent progression of atherosclerosis.

Resveratrol mediates anti-atherogenic effects on cholesterol flux in human macrophages and endothelium via PPARγ and adenosine.

Resveratrol is a bioactive molecule used in dietary supplements and herbal medicines and consumed worldwide. Known cardioprotective and anti-inflammatory properties of resveratrol have spurred investigation of the mechanisms involved. The present study explored potential atheroprotective actions of resveratrol on cholesterol metabolism in cells of the arterial wall, including human macrophages and arterial endothelium. Using QRT-PCR and Western blotting techniques, we measured expression of the proteins involved in reverse cholesterol transport (ABCA1, ABCG1 and SR-B1) and the scavenger receptors responsible for uptake of modified cholesterol (CD36, SR-A1 and LOX-1). We analyzed the effect of resveratrol on apoA-1-and HDL-mediated cholesterol efflux in human THP-1 macrophages. The effect of resveratrol on oxLDL internalization and foam cell formation were evaluated using confocal and light microscopy. Our data indicate that resveratrol regulates expression of major proteins involved in cholesterol transport, promotes apoA-1 and HDL-mediated efflux, downregulates oxLDL uptake and diminishes foam cell formation. Mechanistically, resveratrol effects were dependent upon PPAR-γ and adenosine 2A receptor pathways. For the first time we demonstrate that resveratrol regulates expression of the cholesterol metabolizing enzyme cytochrome P450 27-hydroxylase, providing efficient cholesterol elimination via formation of oxysterols. This study establishes that resveratrol attenuates lipid accumulation in cultured human macrophages via effects on cholesterol transport. Further in vivo studies are needed to determine whether resveratrol may be an additional resource available to reduce lipid deposition and atherosclerosis in humans.

The ABC transporters in lipid flux and atherosclerosis.

Atherosclerotic cardiovascular disease is the leading cause of morbidity and mortality in the United States and in many other countries. Dysfunctional lipid homeostasis plays a central role in the initiation and progression of atherosclerotic lesions. The ATP-binding cassette (ABC) transporters are transmembrane proteins that hydrolyze ATP and use the energy to drive the transport of various molecules across cell membranes. Several ABC transporters play a pivotal role in lipid trafficking. They are critically involved in cholesterol and phospholipid efflux and reverse cholesterol transport (RCT), processes that maintain cellular cholesterol homeostasis and protect arteries from atherosclerosis. In this article we provide a review of the current literature on the biogenesis of ABC transporters and highlight their proposed functions in atheroprotection.

Cholesterol and apolipoprotein E in Alzheimer's disease.

Alzheimers disease (AD) is the most common cause of dementia in North America and Europe. The incidence of the disease rises dramatically with age. AD is a complex multifactorial disorder that involves numerous susceptibility genes, but the exact pathogenesis and biochemical basis of AD is not well understood. Cholesterol is receiving a great deal of attention as a potentially crucial factor in the etiology of AD. Almost all cholesterol in the brain is synthesized in the brain. Cholesterol exits the brain through the blood-brain barrier (BBB) in the form of apolipoprotein E (ApoE) or by first being converted to a more polar compound, 24(S)-hydroxycholesterol, which is elevated in individuals with AD. The key event leading to AD appears to be the formation and aggregation in the brain of amyloid β (Aβ) peptide, a proteolytically derived product of amyloid precursor protein (APP). Cholesterol has been demonstrated to modulate processing of APP to Aβ. High levels of cholesterol are associated with increased risk of AD. Patients taking cholesterol-lowering statins have a lower prevalence of AD. ApoE, which transports cholesterol throughout the brain, exhibits an isoform-specific association with AD such that the E4 isoform, by unknown mechanisms, shifts the onset curve toward an earlier age.

Atherosclerosis: Immune and Inflammatory Aspects

This review article discusses the historical origin of our continuously evolving model of the etiology of atherosclerotic cardiovascular disease. The basic molecular biologic concepts underlying the development of coronary artery disease and the dynamic connection between the immune system and arterial integrity are explored. Emphasis is placed on the role of inflammation as a driving force in the process of atherosclerosis and vascular endothelium as a modulating factor in the pathogenesis of coronary artery disease.&NA; This review article discusses the historical origin of our continuously evolving model of the etiology of atherosclerotic cardiovascular disease. The basic molecular biologic concepts underlying the development of coronary artery disease and the dynamic connection between the immune system and arterial integrity are explored. Emphasis is placed on the role of inflammation as a driving force in the process of atherosclerosis and vascular endothelium as a modulating factor in the pathogenesis of coronary artery disease.

Role of HMG-CoA reductase inhibitors in neurological disorders : progress to date.

Inhibitors of HMG-CoA reductase (statins) are cholesterol-lowering agents that dramatically reduce morbidity and mortality in patients with established cardiovascular disease. In addition, they exhibit pleiotropic effects that operate independently of lipid modification. Statin administration results in greater nitric oxide bioavailability, improved endothelial function, enhanced cerebral blood flow, immune modulation with anti-inflammatory action, decreased platelet aggregation and antioxidant activity. Some or all of these effects may improve outcome or ameliorate symptoms in neurological disorders. This article examines the potential role of statins in treating stroke, Alzheimer’s disease, multiple sclerosis and Parkinson’s disease. Studies are ongoing in this controversial area, but there are no firm conclusions. The appropriateness of initiating statin therapy for neurological disorders is not established at this time. The exception is stroke, in which recurrence is significantly reduced by statin therapy.