Faculty Bibliography

Atherosclerosis is a chronic progressive disease that is a major contributor to cardiac death. It is characterized by inflammation and cholesterol deposition in the arterial wall. Excess cholesterol accumulation occurs as a result of an imbalance between delivery and removal and leads to formation of lipid-laden foam cells. Removal of cholesterol through a process known as reverse cholesterol transport requires the coordinated functioning of a number of genes including the P450 27-hydroxylase and the adenosine triphosphate-binding cassette transporter A1 (ABCA1). Reverse cholesterol transport is a key defense against atheroma formation. This review discusses the role of inflammatory processes in impeding reverse cholesterol transport. Particular emphasis is placed on the disruption of cholesterol outflow observed in the presence of cyclooxygenase inhibitors in cultured monocytes/macrophages. These inhibitors, which are used clinically to relieve pain and inflammation, have been associated with increased risk of cardiovascular disease and myocardial infarction. We explore the relationship between suppression of reverse cholesterol transport and harmful cardiac effects of coxibs

Purpose: Unlike other DMARDs methotrexate diminishes the risk of Atherosclerotic Cardiovascular Disease (ASCVD) in patients with Rheumatoid Arthritis and adenosine, acting at adenosine A2A receptors, has been shown to mediate the anti-inflammatory effects of methotrexate. Adenosine inhibits the first step in formation of atherosclerotic plaque, foam cell formation in macrophages and this effect appears to be mediated by enhanced expression of cholesterol 27-hydroxylase, an enzyme involved in reverse cholesterol transport. We therefore asked whether the effect of adenosine A2A receptors on foam cell formation in vitro are mediated by apoE or 27-hydroxylase (27OH'ase), proteins involved in reverse cholesterol transport. Method: THP-1 cells, a human monocytoid cell line, were infected with lentiviral vectors expressing siRNA for either apoE or 27OH'ase or scrambled RNA and infected cell lines were selected by incubation with puromycin. Foam cell formation was induced in THP-1 cells by incubation with interferon- (500U/ml) and % foam cells enumerated in 5 high power fields. 3H-Cholesterol efflux was measured after loading with label. Results: Specific lentiviral siRNA infection markedly reduces apoE (p< 0.0001, apoE siRNA vs. control, n=3) or 27OH'ase mRNA (p< 0.0001, 27-hydroxylase siRNA vs. control, n=3) and protein (p< 0.0107, 27-hydroxylase siRNA vs. control n= 3) in THP-1 cells. Despite diminished apoE expression CGS-21680 (1muM), an adenosine A2A receptor agonist, inhibits IFN-induced foam cell formation (p< 0.0002, IFN CGS vs. IFN alone, n= 4) but has no effect on foam cell formation in 27OH'ase KD cells. CGS21680 increases cholesterol efflux in wild type and apoE1 KD cells (from 9.5% to 17.5+2.5% and from 10.0+2% to 17.5 +2%, respectively) but not 27OH'ase KD cells. Conclusion: Adenosine A2A receptor-mediated increases in reverse cholesterol transport leading to diminished foam cell formation explains the anti-atherosclerotic effects of methotrexate

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

Both selective cyclooxygenase (COX)-2 inhibitors and non-steroidal anti-inflammatory drugs (NSAIDs) have been beneficial pharmacological agents for many patients suffering from arthritis pain and inflammation. However, selective COX-2 inhibitors and traditional NSAIDs are both associated with heightened risk of myocardial infarction. Possible pro-atherogenic mechanisms of these inhibitors have been suggested, including an imbalance in prostanoid production leaving the pro-aggregatory prostaglandins unopposed, but the precise mechanisms involved have not been elucidated. We explored the possibility that downregulation of proteins involved in reverse cholesterol transport away from atheromatous plaques contributes to increased atherogenesis associated with COX inhibition. The reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) export cholesterol from macrophages. When mechanisms to process lipid load are inadequate, uncontrolled cholesterol deposition in macrophages transforms them into foam cells, a key element of atheromatous plaques. We showed that in cultured THP-1 human monocytes/macrophages, inhibition of COX-1, COX-2, or both reduced expression of 27-hydroxylase and ABCA1 message (real-time reverse transcription-polymerase chain reaction) and protein (immunoblot). The selective COX-2 inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398) significantly reduced 27-hydroxylase and ABCA1 message (to 62.4% +/- 2.2% and 71.1% +/- 3.9% of control, respectively). Incubation with prostaglandin (PG) E2 or PGD2 reversed reductions in both of these cholesterol transport proteins induced by NS398. Cholesterol-loaded THP-1 macrophages showed significantly increased foam cell transformation in the presence of NS398 versus control (42.7% +/- 6.6% versus 20.1% +/- 3.4%, p = 0.04) as determined by oil red O staining. Pharmacological inhibition of COX in monocytes is involved in downregulation of two proteins that mediate cholesterol efflux: cholesterol 27-hydroxylase and ABCA1. Because these proteins are anti-atherogenic, their downregulation may contribute to increased incidence of cardiac events in patients treated with COX inhibitors. Reversal of inhibitory effects on 27-hydroxylase and ABCA1 expression by PGD2 and PGE2 suggests involvement of their respective signaling pathways. NS398-treated THP-1 macrophages show greater vulnerability to form foam cells. Increased cardiovascular risk with COX inhibition may be ascribed at least in part to altered cholesterol metabolism

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

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.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).Adenosine A(2A) receptors are expressed on rat and human hepatic stellate cell lines and adenosine A(2A) receptor occupancy promotes collagen production by these cells. Liver sections from mice treated with the hepatotoxins carbon tetrachloride (CCl(4)) (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.Adenosine A(2A) receptor-deficient, but not wild-type or A(3) receptor-deficient, mice are protected from development of hepatic fibrosis following CCl(4) or thioacetamide exposure.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 A(2A) receptor, diminished hepatic fibrosis in wild-type mice exposed to either CCl(4) or thioacetamide.These results demonstrate that hepatic adenosine A(2A) 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.British Journal of Pharmacology advance online publication, 19 June 2006; doi:10.1038/sj.bjp.0706812