Director, Alain Tedgui
email: alain.tedgui@larib.inserm.fr

The role of inflammation in atherothrombosis: implications for clinical practice

Keywords: cytokines, immunology, inflammation, molecular risk factors, plaque rupture, platelets

The role of inflammation in atherothrombosis:

implications for clinical practice

Introduction

Adverse vascular events such as myocardial infarction (MI), stroke and peripheral arterial disease (PAD) are the most common causes of mortality and morbidity in the Western world today. Epidemiological studies indicate that the prevalence of vascular disease is increasing on a global basis and is likely to reach epidemic proportions in the coming decades. [START_REF] Yusuf | The global epidemic of atherosclerotic cardiovascular disease[END_REF][START_REF] Bonow | World Heart Day 2002: the international burden of cardiovascular disease: responding to the emerging global epidemic[END_REF][START_REF] Lopez | The global burden of disease, 1990-2020[END_REF] Atherothrombosis is the pathological process underlying adverse vascular events and involves the complex sequence of events leading to atherosclerotic plaque development, plaque rupture and thrombus formation. [START_REF] Falk | Coronary plaque disruption[END_REF] It is this final stage in the process that is responsible for clinically observable thrombotic events and adverse vascular outcomes in cardiovascular, cerebrovascular and peripheral vascular beds. Studies indicate that, in patients with atherothrombotic disease, plaque formation is likely to be widespread throughout the vasculature, often affecting more than one vascular bed. [START_REF] Droste | Evaluation of progression and spread of atherothrombosis[END_REF][START_REF] Buffon | Widespread coronary inflammation in unstable angina[END_REF] Over the last few years, increasing evidence has emerged that inflammation plays a key role in the development of atherosclerotic plaques, in plaque rupture and thrombus formation. [START_REF] Libby | Inflammation in atherosclerosis[END_REF][START_REF] Glass | Atherosclerosis: the road ahead[END_REF] As yet, our understanding of these processes is not fully developed, although recent discoveries in vascular biology continue to add to our knowledge base. Nevertheless, as we continue to learn more about the role played by inflammation in the atherothrombotic process, existing knowledge can be applied to the clinical management of cardiovascular disease. [START_REF] Libby | Vascular biology of atherosclerosis: overview and state of the art[END_REF] Future research is likely to increase our understanding of the biochemistry underlying atherothrombosis and will continue to have important implications for current and future therapeutic approaches.

This article seeks to explore recent developments in elucidating the role of inflammation in the development of atherothrombosis and to consider the implications for the clinical management of patients with atherothrombotic disease.

Role of inflammation in the pathogenesis of atherothrombosis

Plaque formation and rupture is a complex process involving many inflammatory cell types and numerous biochemical mediators. The process is a dynamic one with plaque structure and composition responding to the local environment. [START_REF] Lutgens | Atherosclerotic plaque rupture: local or systemic process?[END_REF] Plaques can exist as stable structures or may be unstable and prone to rupture. While stable plaques are relatively harmless, unstable plaques lead to atherothrombotic events and adverse vascular outcomes. [START_REF] Virmani | Pathology of the unstable plaque[END_REF] At the very early stages of plaque formation, inflammation of the endothelium occurs. Lipoprotein accumulation in the arterial wall is observed, probably as a consequence of high circulating levels of cholesterol and oxidation of low-density lipoprotein (LDL).

Recruitment of monocytes and T-lymphocytes follows, mediated by specific leukocyte adhesion molecules expressed on the surface of the vascular cells. Monocytes and T-lymphocytes then penetrate into the arterial intima. [START_REF] Robbie | Inflammation and atherothrombosis[END_REF] This early recruitment of inflammatory cells leads to a complex cascade sequence of expression of inflammatory mediators, which promote the inflammatory process.

The next stage in the process of plaque formation is the differentiation of monocytes into macrophages and the development of foam cells consequent to the ingestion of cholesterol by macrophages. This is followed by migration of smooth muscle cells into the developing plaque and the secretion of collagen, which aids in the formation of a fibrous cap. [START_REF] Ross | Atherosclerosis -an inflammatory disease[END_REF] Atherosclerotic plaques can continue to grow and develop over significant periods of time. Providing the plaque structure remains stable, rupture is unlikely and therefore without significant clinical consequences. [START_REF] Libby | Stabilization of atherosclerotic plaques: new mechanisms and clinical targets[END_REF] However, a shift in balance to increased inflammatory activity can lead to changes in plaque structure that confer instability. [START_REF] Kolodgie | Localization of apoptotic macrophages at the site of plaque rupture in sudden coronary death[END_REF] Development of plaque instability generally involves growth of the lipid core, renewed inflammatory response, macrophage activation, secretion of inflammatory mediators, secretion of matrix metalloproteinases (MMPs) that encourage the degradation of collagen and, finally, weakening of the fibrous cap. [START_REF] Ross | Atherosclerosis -an inflammatory disease[END_REF][START_REF] Hutter | Caspase-3 and tissue factor expression in lipid-rich plaque macrophages: evidence for apoptosis as link between inflammation and atherothrombosis[END_REF] Sudden plaque rupture occurs either as a consequence of rupture of the plaque's fibrous cap or superficial erosion of the intimal surface. Both surface erosion and fibrous cap rupture are generally followed by thrombus formation. [START_REF] Ruberg | Atherothrombosis: plaque instability and thrombogenesis[END_REF] Superficial plaque erosion with consequent exposure of the subendothelial collagen promotes platelet aggregation. Once a thrombus is formed, activated platelets promote monocyte adherence to the underlying endothelium and there is active synthesis by platelets of factors that potentiate inflammation. This process promotes further inflammatory activity contributing to further plaque destabilization. [START_REF] Chyu | The role of inflammation in plaque disruption and thrombosis[END_REF]

Inflammatory markers and mediators involved in plaque development and rupture

A large number of biochemical molecules of various kinds have been shown to be involved in the inflammatory process. [START_REF] Shishehbor | Inflammatory and oxidative markers in atherosclerosis: relationship to outcome[END_REF] These include cytokines, adhesion molecules, CD40/CD40 ligand, metalloproteinases, acute phase proteins, asymmetric dimethylarginine (ADMA), isoprostanes, tissue factor and plasminogen activator inhibitor-1 (PAI-1). Some studies have also demonstrated an association between coronary artery disease and increased plasma levels of oxidized LDL. [START_REF] Holvoet | Oxidative modification of low-density lipoproteins in atherothrombosis[END_REF] Furthermore, antibodies to oxidized LDL have been shown to predict MI and progression of carotid atherosclerosis in non-autoimmune subjects. [START_REF] Vaarala | Antibodies to oxidized LDL[END_REF] Table 1 lists some of the main mediators that have been identified as having an association with plaque development and rupture. For some of these substances the precise role played in the plaque development process has been established, while for others it remains obscure. Some mediators appear to have predominately pro-inflammatory activity, while others are anti-inflammatory in nature. The nature and progress of an atherosclerotic plaque appears to depend upon the prevailing balance of pro-and anti-inflammatory activity. [START_REF] Tedgui | Anti-inflammatory mechanisms in the vascular wall[END_REF]

Cytokines

The cytokine family is probably the most studied group of inflammatory mediating molecules and includes lymphokines (e.g. macrophage activating factor [MAF], macrophage migration inhibition factor [MIF]), interleukins (e.g. IL-1, IL-2, IL-6, IL-10, IL-12, IL-18), tumour necrosis factor (TNF-␣), chemokines (e.g. IL-8, monocyte chemoattractant protein 1 [MCP-1]) and interferon (IFN-␥). Cytokines play a number of different roles in the development of inflammation, including controlling the duration and nature of the immune response and the remodelling that accompanies inflammation. [START_REF] Libby | Cytokines regulate vascular functions related to stability of the atherosclerotic plaque[END_REF] Cytokines exert their effects by facilitating inter-cell signalling. Some cytokines are chemoattractant (e.g. MCP-1) and facilitate the movement of inflammatory cells within the developing plaque; [START_REF] Nelken | Monocyte chemoattractant protein-1 in human atheromatous plaques[END_REF] others stimulate the production of acute phase proteins. [START_REF] Dayer | The process of identifying and understanding cytokines: from basic studies to treating rheumatic disease[END_REF] Animal studies have shown that cytokines such as IL-10 26 and transforming growth factor (TGF-␤) [START_REF] Mallat | Inhibition of transforming growth factor-beta signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice[END_REF] have anti-inflammatory properties and inhibit the production of pro-inflammatory cytokines. Anti-inflammatory cytokines have been shown to have anti-atherogenic properties. [START_REF] Tedgui | Anti-inflammatory mechanisms in the vascular wall[END_REF] TNF-␣ is a potent inflammatory cytokine. TNF receptor 1 and TNF receptor 2 are both soluble receptors shed by the many cell types on which they reside. TNF-␣ promotes the expression of adhesion molecules such as ICAM-1 on the endothelium, facilitating leukocyte adherence and migration from capillaries into the brain, microvessel occlusion, and subsequently a progressive reduction in blood flow. [START_REF] Kapadia | The role of cytokines in the failing human heart[END_REF] Elevation of TNF-␣ and TNF receptor levels occurs in a variety of infectious, inflammatory, autoimmune, and neoplastic diseases. Elevated levels of TNF receptor may be a reflection of the inflammatory mechanisms operative in the atherosclerotic plaque. [START_REF] Castellanos | Inflammationmediated damage in progressing lacunar infarctions: a potential therapeutic target[END_REF] IL-6 is a multifunctional proinflammatory and vasodepressor cytokine that mediates both immune and inflammatory responses. Produced in response to several factors, including IL-1, IFN-␥ and TNFs, it is the primary determinant of C-reactive protein (CRP) release from the liver and the only substance known to induce synthesis of all acute phase proteins. [START_REF] Baumann | Regulation of hepatic acute phase plasma protein genes by hepatocyte stimulating factors and other mediators of inflammation[END_REF] IL-6 gene transcripts are known to be expressed in human atheromatous lesions. [START_REF] Seino | Interleukin 6 gene transcripts are expressed in human atherosclerotic lesions[END_REF] Production of inflammatory markers is stimulated by circulating cytokines such as IL-6 and TNF-␣, which in turn may also be generated from a variety of systemic sources, including adipose tissue, which is a potent source of cytokines, and inflammatory cells either in the atherosclerotic lesion in the arterial wall or elsewhere.

Adhesion molecules

Adhesion molecules shed from a variety of cells give rise to soluble forms measured in the plasma. Adhesion molecules such as ICAM-1 vascular cell ahersion molecule (VCAM-1) and E-selectin are expressed by endothelial cells and play a role in facilitating the contact between inflammatory cells and endothelial cells. [START_REF] Price | Cellular adhesion molecules and atherogenesis[END_REF] Soluble VCAM-1 (sVCAM-1) is another well-defined soluble adhesion molecule which participates actively in the inflammatory-endothelial adhesive interaction process in atherothrombosis. [START_REF] Blan | Circulating endothelial cell/leukocyte adhesion molecules in atherosclerosis[END_REF] Both sVCAM-1 and sICAM-1 have been used as noninvasive markers for the evaluation of endothelial dysfunction.

Available evidence suggests a role for the selectins in the early stages of atherogenesis. E-selectin is synthesized de novo by endothelial cells when activated by IL-1 or TNF-␣. [START_REF] Etter | IL-4 and IL-13 downregulate rolling adhesion of leukocytes to IL-1 or TNF-alpha-activated endothelial cells by limiting the interval of E-selectin expression[END_REF] P-selectin, an adhesion molecule expressed by platelets, facilitates the anchoring of inflammatory cells to the platelet and to endothelial cells. [START_REF] Shebuski | Role of inflammatory mediators in thrombogenesis[END_REF] E-selectin and P-selectin are preferentially expressed in the endothelium overlying atherosclerotic plaques. 36 CD40/CD40 ligand CD40 ligand (CD40L), a key signalling molecule that has many functions in the immune system, is a cell surface molecule and is expressed by activated T-lymphocytes and platelets. CD40 ligand is released by activated platelets during thrombus formation. [START_REF] Samara | The role of platelet receptors and adhesion molecules in coronary artery disease[END_REF] CD40L also exists in a soluble form (sCD40L), which is shed from lymphocytes and platelets within minutes to hours after cell activation. [START_REF] Henn | The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by co-expressed CD40[END_REF] Studies have shown that CD40L can interact with endothelial cells, smooth muscle cells and macrophages to promote plaque instability. [START_REF] Lutgens | CD40-CD40L interactions in atherosclerosis[END_REF] The precise way in which released CD40L affects plaque composition is unclear; however, studies have demonstrated that antibody blockade of CD40L in apoEϪ/Ϫ mice leads to an increase in plaque stability. [START_REF] Schonbeck | Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice[END_REF]

Metalloproteinases

The MMP family of enzymes plays a key role in tissue remodelling. [START_REF] Galis | Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly[END_REF] They can also act as regulatory molecules by processing matrix proteins, cytokines, growth factors and adhesion molecules to generate fragments with enhanced or reduced biological effects. Leukocytes, particularly macrophages, are a major source of MMP production, and MMP release by leukocytes plays a vital role in allowing leukocytes to extravasate and penetrate tissues.

It is proposed that infiltrating inflammatory cells interact with the extracellular matrix and oxidized lipoproteins, contributing to increased production of MMP-9 by macrophages. [START_REF] Xu | Oxidized low-density lipoprotein regulates matrix metalloproteinase-9 and its tissue inhibitor in human monocyte-derived macrophages[END_REF] The presence of increased macrophages provides stimulus for synthesis or activation of MMPs by neighbouring cells facilitating further structural changes and advancement of atherosclerotic lesions. [START_REF] Pasterkamp | Expansive arterial remodeling: location, location, location[END_REF] The MMPs contribute to the development of de novo atherosclerotic plaque as well as to the rupture of these plaques by degrading the associated extracellular matrix.

Acute phase proteins

Acute phase proteins (e.g. CRP, fibrinogen and serum amyloid A) are primarily produced in the liver and are indicative of inflammation at sites distant from the liver. However, recent evidence suggests that CRP may also have a direct role in the pathogenesis of atherosclerotic lesion formation. [START_REF] Ridker | Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men[END_REF][START_REF] Jialal | C-reactive protein: risk marker or mediator in atherothrombosis?[END_REF] CRP appears to affect a number of interrelated pathways in the vascular endothelium, including the induction of expression of adhesion molecules, foam cell formation and monocyte recruitment in the arterial wall. [START_REF] Ridker | Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men[END_REF] Blood viscosity depends mainly on the amount of blood cell content and plasma proteins, mainly fibrinogen. Thrombin converts fibrinogen into fibrin, which, after polymerization, converts into crosslinking fibrin. The presence of fibrin monomer is considered to indicate a prothrombotic state. [START_REF] Saigo | Soluble fibrin, C-reactive protein, fibrinogen, factor VII, antithrombin, proteins C and S, tissue factor, D-dimer, and prothrombin fragment 1ϩ2 in men with acute myocardial infarction. Ͻ/ϭ45 years of age[END_REF]

ADMA

ADMA is an endogenous inhibitor of nitric oxide (NO) synthase, a crucial enzyme in the maintenance of vascular tone and structure. Ito et al. [START_REF] Ito | Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase[END_REF] showed that incubation of cultured human endothelial cells with oxidized LDL or TNF-␣ increased the level of ADMA in the conditioned medium. ADMA is metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), and inhibition of DDAH activity has been shown to lead to increased ADMA levels and endothelial dysfunction.

Isoprostanes

Isoprostanes are a complex family of compounds produced from arachidonic acid via a free-radical-catalysed mechanism. F 2 -isoprostanes are prostaglandin-F 2 -like compounds generated by free radical-induced peroxidation of arachidonic acid [START_REF] Morrow | A series of prostaglandin F 2 -like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism[END_REF] and are used extensively as clinical markers of lipid peroxidation in cardiovascular disorders. Lipid oxidation has been shown to be a prominent feature of atherosclerosis. The presence of isoprostanes and other lipid oxidation products has been clearly demonstrated in lesions. [START_REF] Witztum | Oxidized phospholipids and isoprostanes in atherosclerosis[END_REF] Quantification of isoprostanes provides a non-invasive approach for studying lipid peroxidation in vivo.

Tissue factor

Tissue factor is released in the bloodstream once an atherothrombotic plaque is disrupted, triggering the formation of a superimposed thrombus that leads to vessel occlusion and subsequent ischemic symptoms distal to it. [START_REF] Fuster | Epidemic of cardiovascular disease and stroke: the three main challenges. Presented at the 71st scientific sessions of the American Heart Association[END_REF] Specific inhibition of the tissue factor pathway by its physiologic inhibitor, tissue factor pathway inhibitor (TFPI), significantly reduces plaque thrombogenicity. [START_REF] Badimon | Local inhibition of tissue factor reduces the thrombogenicity of disrupted human atherosclerotic plaques: effects of tissue factor pathway inhibitor on plaque thrombogenicity under flow conditions[END_REF] PAI-1 PAI-1 is a serine protease inhibitor whose major function is to negate plasminogen activation and impair fibrinolysis. [START_REF] Alessi | Contribution of PAI-1 in cardiovascular pathology[END_REF] It is present in plasma and tissues. Circulating PAI-1 levels are thought to play an important role in the development of atherothrombosis by decreasing fibrin degradation. It may be that increased PAI-1 in blood might facilitate persistence of microthrombi and a prothrombotic state, resulting in attenuation of thickness of elaborated fibrous caps implicated in the vulnerability of atheroma and rupture.

Mediators and markers as predictors of cardiovascular risk

Increased expression of many of these inflammatory substances has been shown to be correlated with an increased risk of atherothrombotic events in a number of studies. Increased expression can be identified as either an elevated local concentration of a substance or raised systemic levels. Substances that have a clear role in the inflammatory response can be regarded both as mediators and markers of inflammation. Substances with an established relationship between increased expression and increased cardiovascular risk but an unclear basis of correlation should be regarded only as markers for inflammation.

Correlation between systemic levels of a substance and the development of vascular disease has important clinical implications as measurement of these substances may be a useful means of predicting cardiovascular risk. Accurate prediction of cardiovascular risk has the dual benefit of providing the physician with a means of assessing a patient's pharmacological requirements and assessing the response to therapy.

A clear correlation between elevated plasma levels and increased risk of vascular disease has been demonstrated for CRP in males, [START_REF] Ridker | Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men[END_REF] CD40 ligand, [START_REF] Heeschen | Soluble CD40 ligand in acute coronary syndromes[END_REF] E-selectin, [START_REF] Hwang | Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases: the Atherosclerosis Risk In Communities (ARIC) study[END_REF] IL-6, [START_REF] Ridker | Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men[END_REF] TNF-␣ [START_REF] Ridker | Elevation of tumor necrosis factor-alpha and increased risk of recurrent coronary events after myocardial infarction[END_REF] and ICAM-1. [START_REF] Blake | Novel clinical markers of vascular wall inflammation[END_REF] Of the substances shown to correlate with vascular disease risk, CRP has been the most widely studied, despite the fact that the relationship between the elevation of circulating levels of CRP and increased risk remains poorly understood.

The fact that plasma concentration of CRP predicts the risk of future MI and stroke was identified in a study in men as long ago as 1997 [START_REF] Ridker | Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men[END_REF] and subsequent studies in men and women have confirmed the correlation between cardiovascular risk and CRP levels. [START_REF] Ridker | Plasma concentration of C-reactive protein and risk of developing peripheral vascular disease[END_REF][START_REF] Kuller | Relation of C-reactive protein and coronary heart disease in the MRFIT nested case-control study. Multiple Risk Factor Intervention Trial[END_REF] Elevated baseline CRP levels have also been shown to predict a heightened risk of 30-day death or MI after coronary intervention. [START_REF] Chew | Incremental prognostic value of elevated baseline C-reactive protein among established markers of risk in percutaneous coronary intervention[END_REF] Recently, the relationship between CRP and the Framingham Coronary Heart Disease Risk Score (FCRS) has been studied in a cross-sectional survey of 1666 individuals free of cardiovascular disease. [START_REF] Albert | Plasma concentration of C-reactive protein and the calculated Framingham Coronary Heart Disease Risk Score[END_REF] Among men and women not using hormone replacement therapy (HRT), CRP levels were significantly related to 10-year FCRS categories although correlated only minimally with individual components of the FCRS. Surprisingly, for women taking HRT, no significant relationship was observed between CRP and the FCRS, although the power to detect effects in this subgroup was considered by the investigators to be limited.

Circulating CRP levels are affected by a number of factors associated with cardiovascular disease, including demographic parameters, lifestyle and pharmacological therapy. CRP levels are decreased by weight loss, exercise and therapies such as lipid-lowering drugs and antiplatelet agents, while CRP levels are increased by smoking, diabetes, elevated triglyceride levels and high blood pressure. [START_REF] Pearson | Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association[END_REF] It remains unclear, however, whether the relationship between increased circulating CRP levels and increased cardiovascular risk is a causal or casual relationship. Furthermore, CRP levels are also influenced by chronic fatigue, coffee consumption, sleep disturbances and depression, which are not traditionally considered to have an influence on cardiovascular risk. Therefore, while it is comparatively simple to make risk predictions based on CRP levels on a population basis, it may be more difficult to interpret CRP levels in the individual patient.

The American Heart Association/Centers for Disease Control (AHA/CDC) workshop assessed the value of various markers for routine risk assessment. [START_REF] Pearson | Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association[END_REF] A recent clinical study, published after the workshop, reported that the CRP concentration is a relatively moderate predictor of the risk of coronary heart disease and adds only marginally to the predictive value of established risk factors for coronary heart disease [START_REF] Danesh | C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease[END_REF] and similar overall findings were observed in an updated meta-analysis involving a total of 7068 patients with coronary heart disease, [START_REF] Danesh | C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease[END_REF] suggesting that CRP may be a relatively moderate predictor of coronary heart disease. It must be noted though that the clinical study mentioned above involved patients from The Reykjavik Study, which reflects an Icelandic population. Therefore, although the relative homogeneity of the Reykjavik population should have minimized certain residual biases, further clarification of the predictive value of CRP in coronary heart disease in the general population will require pooling studies on the basis of data for individual participants from each of the available prospective studies.

The participants of the AHA/CDC workshop concluded that to be useful in regular clinical practice, a marker would have to be independent from established risk factors, be clearly correlated with clinical endpoints with data available on normal population values, improve the overall risk prediction beyond traditional factors, be a generalization across various populations and have an assay that could be standardized with an acceptable cost. Of the currently available assays for inflammatory markers, only the high sensitivity assay for CRP (hsCRP) was considered to meet these criteria. The participants recommended that relative risk be categorized as low, average or high corresponding to approximate tertiles of values: Ͻ1.0, 1.0-3.0 and Ͼ3.0 mg/l, respectively. However, the workshop also asserted that, for primary prevention of coronary disease, CRP should only be used as an adjunct to the major risk factors to further assess absolute risk, while, for secondary prevention, intensive treatment was required regardless of CRP levels.

TNF-␣ has also been associated with an elevated risk of recurrent MI and cardiovascular death after a first MI. [START_REF] Ridker | Elevation of tumor necrosis factor-alpha and increased risk of recurrent coronary events after myocardial infarction[END_REF] TNF-␣ levels were correlated with the ankle-brachial index. [START_REF] Bruunsgaard | A high plasma concentration of TNFalpha is associated with dementia in centenarians[END_REF] Other investigators have suggested that soluble TNF receptor levels may be a better marker of atherosclerotic burden than TNF-␣ itself. A study that sought to determine whether TNF-␣ and TNF receptor levels were associated with carotid plaque thickness concluded that relative elevation in TNF receptor levels, but not TNF-␣, was associated with carotid atherosclerosis among individuals aged Ͻ70 years, suggesting that TNF receptors may be a more stable marker of inflammatory burden than TNF-␣. [START_REF] Castellanos | Inflammationmediated damage in progressing lacunar infarctions: a potential therapeutic target[END_REF] Other inflammatory markers have also been related to cardiovascular risk. Among apparently healthy men in the Physicians' Health study, baseline levels of IL-6 were independently predictive of risk of future MI. [START_REF] Ridker | Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men[END_REF] Soluble ICAM-1, known to mediate leukocyte adhesion and transmigration across the vascular wall, has been found to be higher in patients with stable angina, unstable angina, and acute MI compared with control patients. [START_REF] Haught | Alterations in circulating intercellular adhesion molecule-1 and L-selectin: further evidence for chronic inflammation in ischemic heart disease[END_REF] Higher levels of E-selectin have been found with elevations of ICAM-1 in patients with either coronary heart disease or carotid artery atherosclerosis compared with control patients. [START_REF] Hwang | Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases: the Atherosclerosis Risk In Communities (ARIC) study[END_REF] In a small study, soluble fibrin monomer complex was related to the development of MI in young patients. [START_REF] Saigo | Soluble fibrin, C-reactive protein, fibrinogen, factor VII, antithrombin, proteins C and S, tissue factor, D-dimer, and prothrombin fragment 1ϩ2 in men with acute myocardial infarction. Ͻ/ϭ45 years of age[END_REF] Therefore, fibrin may be a candidate for an early marker for thrombosis, but further studies are needed to evaluate the contribution of this factor to cardiovascular disease. Elevated blood levels of sCD40L appear to be associated with the risk of future cardiovascular events in otherwise healthy women and predict patients with high-risk atherosclerotic lesions. [START_REF] Schönbeck | Soluble CD40L and cardiovascular risk in women[END_REF][START_REF] Blake | Soluble CD40 ligand levels indicate lipid accumulation in carotid atheroma: an in vivo study with high-resolution MRI[END_REF] High plasma levels of fibrinogen are also associated with increased risk of cardiovascular disease. [START_REF] Danesh | Association of fibrinogen, C-reactive protein, albumin, or leukocyte count with coronary heart disease: meta-analysis of prospective studies[END_REF] In the Caerphilly and Speedwell Collaborative Heart Disease Studies, fibrinogen, plasma viscosity and white blood cell count were independent risk factors for ischemic heart disease. [START_REF] Yarnell | Fibrinogen, viscosity, and white blood cell count are major risk factors for ischaemic heart disease: the Caerphilly and Speedwell Collaborative Heart Disease Studies[END_REF] ADMA has also been shown to be increased in the plasma of humans with hypercholesterolemia or atherosclerosis. A study assessing the progression of the intima-media thickness during a period of 1 year found that ADMA and CRP levels emerged as the sole independent predictors of the progression of intimal lesions in patients with initially normal intima-media thickness. [START_REF] Zoccali | Asymmetric dimethylarginine (ADMA), C-reactive protein, and carotid intima media-thickness in end-stage renal disease[END_REF] A prospective, nested, case-control study has demonstrated that middle-aged men who did not smoke, but who had ADMA plasma levels in the highest quartile, had a 3.9-fold increased risk for acute coronary events compared with the other quartiles. [START_REF] Valkonen | Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine[END_REF] Further trials are warranted to prove the hypothesis that ADMA is a predictor of cardiovascular events and/or mortality, and thus to assess its suitability as a diagnostic marker of ischemic vascular disease.

Isoprostanes have been detected in human atherosclerotic plaque [START_REF] Pratico | Localization of distinct F2-isoprostanes in human atherosclerotic lesions[END_REF] and levels of isoeicosanoids, free radical-catalysed isomers of the traditional enzymatic products of arachidonic acid, are higher in patients with symptomatic plaques than in their asymptomatic peers, [START_REF] Mallat | The relationship of hydroxyeicosatetraenoic acids and F2-isoprostanes to plaque instability in human carotid atherosclerosis[END_REF] suggesting an association with plaque instability.

Circulating tissue factor has been associated with increased blood thrombogenicity in patients with unstable angina and chronic coronary artery disease, [START_REF] Kaikita | Tissue factor expression on macrophages in coronary plaques in patients with unstable angina[END_REF] and there is evidence that acute thrombosis may be initiated by membrane-bound circulating tissue factor originating from activated or injured cells. [START_REF] Giesen | Blood-borne tissue factor: another view of thrombosis[END_REF] Elevated levels of PAI-1 have been linked to increased cardiovascular risk in middle-aged men [START_REF] Schönbeck | Soluble CD40L and cardiovascular risk in women[END_REF] and circulating PAI-1 concentrations are elevated in young men at increased risk for recurrent infarction. [START_REF] Alessi | Contribution of PAI-1 in cardiovascular pathology[END_REF] In the Physicians' Health Study, [START_REF] Ridker | Endogenous tissue-type plasminogen activator and risk of myocardial infarction[END_REF] it appeared that increased EPA activity was associated with increased coronary events.

Implications for therapy

The recent upsurge in our understanding of the role played by inflammation in the atherothrombotic process has significant implications for current and future therapeutic approaches to the primary and secondary prevention of atherothrombotic events.

There is evidence that drugs currently employed for primary and secondary prevention of cardiovascular disease may have anti-inflammatory effects over and above their primary pharmacological effects. Drugs such as ACE inhibitors [START_REF] Scholkens | ACE inhibition and atherogenesis[END_REF] and statins [START_REF] Ridker | Rapid reduction in C-reactive protein with cerivastatin among 785 patients with primary hypercholesterolemia[END_REF] appear to exert beneficial effects even in patients with normal blood pressure or lipid levels. These observations have led to the suggestion that these drug classes possess therapeutic benefits beyond their antihypertensive or lipidlowering effects.

Evidence that statins may modulate inflammatory processes has been derived from studies that have shown that statins can decrease expression of IL-6, IL-8, MCP-1, CRP, TNF-␣ and MMP. [START_REF] Rezaie-Majd | Simvastatin reduces expression of cytokines interleukin-6, interleukin-8, and monocyte chemoattractant protein-1 in circulating monocytes from hypercholesterolemic patients[END_REF][START_REF] Koh | Comparative effects of diet and statin on NO bioactivity and matrix metalloproteinases in hypercholesterolemic patients with coronary artery disease[END_REF] Statins have also been shown to prevent the activation of monocytes into macrophages and inhibit the production of cellular adhesion molecules. [START_REF] Koh | Effects of statins on vascular wall: vasomotor function, inflammation, and plaque stability[END_REF] Similarly, the antidiabetic agent rosiglitazone has been shown to reduce circulating levels of inflammatory markers, suggesting an anti-inflammatory effect of the drug over and above its key pharmacological effect. [START_REF] Haffner | Effect of rosiglitazone treatment on nontraditional markers of cardiovascular disease in patients with type 2 diabetes mellitus[END_REF] Antiplatelet agents have also been shown to have the potential to modify inflammatory processes. Ex vivo studies have demonstrated that clopidogrel treatment, with or without acetylsalicylic acid (ASA), abolishes the ADP-induced expression of CD40L in human platelets. [START_REF] Hermann | Platelet CD40 ligand (CD40L)-subcellular localization, regulation of expression, and inhibition by clopidogrel[END_REF] Clopidogrel (but not ASA) has also been shown to reduce P-selectin expression and formation of platelet-leukocyte aggregates in patients with atherothrombotic disease [START_REF] Klinkhardt | Clopidogrel but not aspirin reduces P-selectin expression and formation of platelet-leukocyte aggregates in patients with atherosclerotic vascular disease[END_REF] and in patients with an acute coronary syndrome. [START_REF] Xiao | Clopidogrel inhibits platelet-leukocyte interactions and thrombin receptor agonist peptide-induced platelet activation in patients with an acute coronary syndrome[END_REF] Furthermore, CRP levels have been shown to be significantly lower 7 days after the event in acute ischaemic stroke patients treated with clopidogrel compared with those receiving heparin treatment, [START_REF] Cha | Changes in platelet p-selectin and in plasma C-reactive protein in acute atherosclerotic ischemic stroke treated with a loading dose of clopidogrel[END_REF] while clopidogrel pretreatment prior to percutaneous coronary intervention (PCI) has also been shown to attenuate the periprocedural rise in CRP. [START_REF] Vivekananathan | Effect of clopidogrel pretreatment on periprocedural rise of C-reactive protein after percutaneous coronary intervention[END_REF] Other anti-inflammatory activities associated with clopidogrel treatment include decreased serum levels of soluble ICAM-1 seen after treatment of healthy volunteers with the drug [START_REF] Dunzendorfer | Inhibition of plasma-dependent monocyte chemokinesis and cytokinetriggered endothelial activation for neutrophil transmigration by administration of clopidogrel in man[END_REF] and the reduced periprocedural expression of P-selectin in PCI patients pretreated with clopidogrel. [START_REF] Quinn | Effect of clopidogrel pretreatment on inflammatory marker expression in patients undergoing percutaneous coronary intervention[END_REF] However, it is unclear whether these anti-inflammatory actions play a significant role in the reduction of cardiovascular risk that these therapies afford and further studies are required to explore these interesting observations. No currently available therapies have convincingly demonstrated a cardiovascular protective effect on the basis of their anti-inflammatory activity.

In the future, the development of new therapies designed for the primary and secondary prevention of atherothrombotic events is likely to focus on strategies that decrease the inflammatory response and tip the balance in favour of anti-inflammatory mediators and, therefore, plaque stability. Approaches may include the development of agents that block proinflammatory cytokine signalling, agents that augment the anti-inflammatory activity of other cytokines and agents that either block the transcription of inflammatory mediating molecules or upregulate anti-inflammatory molecules.

Conclusion

As more is discovered about the complex sequence of inflammatory events involved in plaque formation and rupture, vascular medicine will reap the benefits. Future research should enable improved risk assessment and the development of new therapeutic strategies that target the biochemical perturbations that underlie atherothrombosis.

Inflammation in atherothrombosis 47 Vascular

 47 Medicine 2005; 10: 45-53

Table 1

 1 Pro-and anti-inflammatory markers and mediators involved in plaque development and rupture.

	Marker/mediator	Localization/expression	Physiological function/role
	Cytokines: e.g. IL-1, IL-6,	Macrophages, Th1 lymphocytes	Control duration and nature of immune
	IL-8, IL-12, IL-18, TNF-␣,		response and remodelling that
	IFN-␥ (pro-inflammatory)		accompanies inflammation by inter-
			immune cell signalling; effects may be
			pro-or anti-inflammatory
	IL-10, TGF-␤ (anti-	Macrophages, Th2/Th3
	inflammatory)	lymphocytes
	Adhesion molecules:	ICAM-1, VCAM-1 and E-selectin are	Mediate contact of inflammatory cells with
	e.g. ICAM-1, VCAM-1,	expressed by endothelial cells;	other cells (e.g. endothelial cells)
	P-selectin, E-selectin	P-selectin is expressed by both
		endothelial cells and by platelets
	CD40/CD40 ligand	T-lymphocytes and platelets	Multifunctional signalling molecule
	Metalloproteinases	Macrophages, smooth muscle cells	Tissue remodelling
	Acute phase proteins:	Synthesized in the liver; some	Multiple effects (e.g. induction of adhesion
	e.g. CRP, fibrinogen,	evidence for local vascular	molecule expression)
	serum amyloid A	synthesis of CRP
	ADMA	Inhibitor of nitric oxide synthase	Increased levels associated with
			endothelial dysfunction
	Isoprostanes	Synthesized by free radical-induced	Markers of lipid peroxidation
		peroxidation of arachidonic acid
	Tissue factor	Present in atherosclerotic plaques	Involved in plaque thrombogenicity
	PAI-1	Serine protease inhibitor	Impairs fibrinolysis

IL, interleukin; TNF-␣, tumour necrosis factor alpha; IFN-␥, interferon gamma; TGF-␤, transforming growth factor beta; ICAM, intercellular adhesion moelcule; VCAM, vascular cell adhesion molecule. CRP, C-reactive protein; ADMA, asymmetric diemthylarginine; PAI-1, plasminogen activator inhibtor-1.

Vascular Medicine 2005; 10: 45-53

Inflammation in atherothrombosis 49Vascular Medicine 2005; 10:[START_REF] Jialal | C-reactive protein: risk marker or mediator in atherothrombosis?[END_REF][START_REF] Saigo | Soluble fibrin, C-reactive protein, fibrinogen, factor VII, antithrombin, proteins C and S, tissue factor, D-dimer, and prothrombin fragment 1ϩ2 in men with acute myocardial infarction. Ͻ/ϭ45 years of age[END_REF][START_REF] Ito | Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase[END_REF][START_REF] Morrow | A series of prostaglandin F 2 -like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism[END_REF][START_REF] Witztum | Oxidized phospholipids and isoprostanes in atherosclerosis[END_REF][START_REF] Fuster | Epidemic of cardiovascular disease and stroke: the three main challenges. Presented at the 71st scientific sessions of the American Heart Association[END_REF][START_REF] Badimon | Local inhibition of tissue factor reduces the thrombogenicity of disrupted human atherosclerotic plaques: effects of tissue factor pathway inhibitor on plaque thrombogenicity under flow conditions[END_REF][START_REF] Alessi | Contribution of PAI-1 in cardiovascular pathology[END_REF][START_REF] Ridker | Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men[END_REF]

Acknowledgement

Alain Tedgui is an advisor to Sanofi-Aventis and Bristol-Myers Squibb.

Note in proof

After this manuscript was accepted for publication, two articles published in the January 6, 2005 issue of the New England Journal of Medicine, one by Nissen et al. and one by Ridker et al., confirmed that reducing CRP levels through the use of statin therapy improves the clinical outcome independently of the reduction in serum LDL cholesterol levels. [START_REF] Nissen | Effects of statin therapy on LDL cholesterol, C-reactive protein, and the progression of coronary artery disease[END_REF][START_REF] Ridker | Clinical relevance of C-reactive protein levels after statin therapy[END_REF]


Soni Soumian
email: s.soumian@imperial.ac.uk

C Albrecht

A H Davies

Rgj Gibbs

ABCA1 and atherosclerosis

Keywords: apolipoprotein A1, atherosclerosis, ATP binding cassette transporter 1, HDL cholesterol, Tangier disease

ATP binding cassette transporter A1 (ABCA1) mediates the cellular efflux of phospholipids and cholesterol to lipid-poor apolipoprotein A1 (apoA1) and plays a significant role in high density lipoprotein (HDL) metabolism. ABCA1's role in the causation of Tangier disease, characterized by absent HDL and premature atherosclerosis, has implicated this transporter and its regulators liver-X-receptor␣ (LXR␣) and peroxisome proliferator activated receptor␥ (PPAR␥) as new candidates potentially influencing the progression of atherosclerosis. In addition to lipid regulation, these genes are involved in apoptosis and inflammation, processes thought to be central to atherosclerotic plaque progression. A Medline-based review of the literature was carried out. Tangier disease and human heterozygotes with ABCA1 mutations provide good evidence that ABCA1 is a major candidate influencing atherosclerosis. Animal and in vitro experiments suggest that ABCA1 not only mediates cholesterol and phospholipid efflux, but is also involved in the regulation of apoptosis and inflammation. The complex and beneficial interactions between apoA1 and ABCA1 seem to be pivotal for cholesterol efflux. The expression of the ABCA1 is tightly regulated. Furthermore the plaque microenvironment could potentially promote ABCA1 protein degradation thus compromising cholesterol efflux. PPAR-LXR-ABCA1 interactions are integral to cholesterol homeostasis and these nuclear receptors have proven anti-inflammatory and anti-matrix metalloproteinase activity. Therapeutic manipulation of the ABCA1 transporter is feasible using PPAR and LXR agonists. PPAR agonists like glitazones and ABCA1 protein stabilization could potentially modify the clinical progression of atherosclerotic lesions.

Introduction

Atherosclerosis, a multifactorial disease of great complexity, starts in fetal life, progresses slowly through childhood and adolescence and accelerates in adult life. The characteristic component of the atherosclerotic plaque is the macrophage derived foam cell. [START_REF] Tabas | Cholesterol and phospholipid metabolism in macrophages[END_REF] This cholesterol laden macrophage is thought to result from the uptake of oxidatively modified low density lipoprotein (LDL). [START_REF] Steinberg | Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity[END_REF] However, intracellular free cholesterol (FC) can be toxic to the cell [START_REF] Simons | How cells handle cholesterol[END_REF] and therefore an efficient cholesterol efflux mechanism in the macrophage is mandatory to prevent cholesterol accumulation. High density lipoprotein (HDL) and its apolipoproteins, especially apolipoprotein A1 (apoA1), are responsible for the transfer of cholesterol from peripheral cells to the liver for biliary excretion, the process of reverse cholesterol transport. [START_REF] Fielding | Molecular physiology of reverse cholesterol transport[END_REF] ATP binding cassette transporter A1 (ABCA1), a membrane transporter abundant in macrophages, mediates this cholesterol and phospholipid (PL) efflux to lipidpoor apoA1, the precursor of HDL, and plays a major role in cholesterol homeostasis and reverse cholesterol transport. [START_REF] Oram | ABCA1: The gatekeeper for eliminating excess tissue cholesterol[END_REF] The various functions of ABCA1 became apparent after the discovery in 1999 that mutations in the ABCA1 gene caused Tangier disease (TD), an autosomal recessive hereditary disorder characterized by severe HDL deficiency, sterol deposition in macrophages and premature atherosclerosis. [START_REF] Bodzioch | The gene encoding ATPbinding cassette transporter 1 is mutated in Tangier disease[END_REF] TD patients with homozygote mutations in the ABCA1 gene have virtually absent HDL and apoA1, decreased low density lipoprotein (LDL) level (40% normal) and hypertriglyceridemia. [START_REF] Oram | Tangier disease and ABCA1[END_REF] The association between ABCA1 and HDL is ascertained by the fact that reduction in ABCA1 activity is associated with a significant decrease in plasma HDL levels. [START_REF] Clee | Age and residual cholesterol efflux affect HDL cholesterol levels and coronary artery disease in ABCA1 heterozygotes[END_REF] Apart from its role in lipid metabolism, ABCA1 has also been implicated in promoting engulfment of apoptotic cells, [START_REF] Hamon | ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine[END_REF] LDL oxidation [START_REF] Reddy | ATP-binding cassette transporter 1 participates in LDL oxidation by artery wall cells[END_REF] and the release of inflammatory mediators. [START_REF] Hamon | Interleukin-1beta secretion is impaired by inhibitors of the ATP binding cassette transporter, ABC1[END_REF][START_REF] Zhou | The ATP binding cassette transporter A1 contributes to the secretion of interleukin 1beta from macrophages but not from monocytes[END_REF] ABCA1 also binds to other lipoproteins including apolipoprotein E thus playing a role in its anti-atherogenic effect. [START_REF] Remaley | Apolipoprotein specificity for lipid efflux by the human ABCAI transporter[END_REF] Atherosclerotic plaque composition is the major determinant of plaque disruption and the ensuing complications. The aetio-pathogenesis of an atheromatous plaque is influenced by lipid metabolism, disordered cell turnover and extracellular matrix turnover within its structure. ABCA1 plays a role in all of these events, thus potentially implicating this transporter in the initiation, progression and pathogenesis of atherosclerotic vascular disease (Figure 1). In this review we will focus on the potential role of ABCA1 in atherosclerotic vascular disease.

Structure and distribution

ABCA1 is a member of the superfamily of ABC transporters that utilize ATP as a source of energy to transport various molecules across membranes. [START_REF] Dean | The human ATP-binding cassette (ABC) transporter superfamily[END_REF] The ABCA1 gene consists of 50 exons spanning 149 kb and encodes a protein that contains 2261 amino acids. [START_REF] Santamarina-Fojo | Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter[END_REF] Immunofluorescence studies suggest that ABCA1 localises in the plasma membrane [START_REF] Wang | Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1[END_REF] although shuttling of ABCA1 between intracellular endocytic components and the plasma membrane has been reported. [START_REF] Neufeld | Cellular localization and trafficking of the human ABCA1 transporter[END_REF] The structure of ABCA1 is characterized by the presence of two transmembrane domains with six helices each and a nucleotide binding domain containing two conserved peptide motifs (Walker A and B) that are characteristic for the superfamily of ABC transporters. It also has two large extracellular loops joined by a disulfide linkage which are thought to be important for the binding of apoA1. [START_REF] Fitzgerald | Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I[END_REF] ABCA1 has been shown to be highly expressed in macrophages. [START_REF] Langmann | Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages[END_REF][START_REF] Lawn | Localization of human ATP-binding cassette transporter 1 (ABC1) in normal and atherosclerotic tissues[END_REF] Though a near ubiquitous tissue expression has been proposed, Wellington et al [START_REF] Wellington | ABCA1 mRNA and protein distribution patterns predict multiple different roles and levels of regulation[END_REF] have shown a significant discordance between relative mRNA and protein expression patterns of ABCA1 in murine tissues.

Mechanism of cholesterol efflux

Cholesterol efflux occurs through different mechanisms, ABCA1 being one of them. The efflux of FC promoted by ABCA1 is considered to be unidirectional and by active transport to apoA1. It is suggested that the golgi apparatus processes excess intracellular FC into vesicles that are translocated to plasma membrane ABCA1 for exocytosis. [START_REF] Zha | Secretory vesicular transport from the golgi is altered during ATP-binding cassette protein A1 (ABCA1)-mediated cholesterol efflux[END_REF] Recent literature suggests that ABCA1 mediates concurrent transport of FC and PL to apolipoproteins [START_REF] Smith | ABCA1 mediates concurrent cholesterol and phospholipid efflux to apolipoprotein A-I[END_REF] though the availability of different lipids in the vicinity of ABCA1 may result in modification of the ratio of cholesterol/phospholipid undergoing efflux. [START_REF] Wang | Regulation and mechanisms of ATP-binding cassette transporter A1-mediated cellular cholesterol efflux[END_REF] It also has been recently shown in transgenic mice that in vivo modification of the PL/apoA1 ratio by various lipases could play a major role in directing FC efflux through either the ABCA1 or the scavenger receptor B1 (SR-B1) pathway. 25

ApoA1 and ABCA1

It is well established that free apolipoproteins play a role in mediating removal of cellular cholesterol. [START_REF] Oram | Apolipoprotein-mediated removal of cellular cholesterol and phospholipids[END_REF] ApoA1 is the most abundant apolipoprotein in HDL and is known to play a role in cholesterol efflux through ABCA1 dependent [START_REF] Chroni | Cross-linking and lipid efflux properties of ApoA-I mutants suggest direct association between ApoA-I helices and ABCA1[END_REF] and independent mechanisms. [START_REF] Walter | The establishment of telomerase-immortalized Tangier disease cell lines indicates the existence of an apolipoprotein A-I-inducible but ABCA1-independent cholesterol efflux pathway[END_REF] To date no clear consensus exists regarding the process by which apoA1 removes cholesterol and PL from the plasma membrane. There are studies supporting the theory of membrane solubilization wherein apoA1 and ABCA1 simultaneously remove both PL and cholesterol through a single step process. [START_REF] Gillotte | Apolipoproteinmediated plasma membrane microsolubilization. Role of lipid affinity and membrane penetration in the efflux of cellular cholesterol and phospholipid[END_REF] Another proposed mechanism includes a two step process in which ABCA1 lipidates apoA1 with PL first to form phospholipid rich nascent HDL particles which then remove cholesterol from cells by diffusion. [START_REF] Fielding | A two-step mechanism for free cholesterol and phospholipid efflux from human vascular cells to apolipoprotein A-1[END_REF][START_REF] Wang | ATP-binding cassette transporter A1 (ABCA1) functions as a cholesterol efflux regulatory protein[END_REF] The flopping of phosphatidylserine (PS) from the inner to the outer leaflet which is promoted by ABCA1 was also thought to play a role in apoA1 mediated lipid efflux [START_REF] Chambenoit | Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter[END_REF] although a recent study suggests that the small increase in PS translocation caused by ABCA1 might be insufficient for apoA1 binding and lipid efflux. [START_REF] Smith | Evaluation of the role of phosphatidylserine translocase activity in ABCA1mediated lipid efflux[END_REF] There is increasing evidence that apoA1 directly interacts with ABCA1. [START_REF] Chroni | Cross-linking and lipid efflux properties of ApoA-I mutants suggest direct association between ApoA-I helices and ABCA1[END_REF][START_REF] Fitzgerald | ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux[END_REF] Further proof of this are studies which showed that mutations in the extracellular loops of ABCA1 impaired both cross-linking with apoA1 and lipid efflux. [START_REF] Fitzgerald | Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I[END_REF] Moreover, truncation mutations of apoA1 lacking helix 10 impaired cholesterol transport through the ABCA1 pathway. [START_REF] Panagotopulos | The role of apolipoprotein A-I helix 10 in apolipoprotein-mediated cholesterol efflux via the ATP-binding cassette transporter ABCA1[END_REF] For efficient apoA1 mediated transport, a functional ABCA1 protein and specific binding at the plasma membrane [START_REF] Chambenoit | Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter[END_REF] is crucial as mutated ABCA1 seen in diseases like TD [START_REF] Bodzioch | The gene encoding ATPbinding cassette transporter 1 is mutated in Tangier disease[END_REF] and familial HDL deficiency, [START_REF] Marcil | Mutations in the ABC1 gene in familial HDL deficiency with defective cholesterol efflux[END_REF] can result in increased catabolism of apoA1 thus affecting HDL production. [START_REF] Francis | Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier disease[END_REF] Animal studies have revealed that apoA1 itself, independent of HDL cholesterol, has properties that protect against atherosclerosis. [START_REF] Moore | Apolipoprotein A-I deficiency results in markedly increased atherosclerosis in mice lacking the LDL receptor[END_REF] ApoA1 has documented roles in inhibiting LDL oxidation, [START_REF] Reddy | ATP-binding cassette transporter 1 participates in LDL oxidation by artery wall cells[END_REF][START_REF] Navab | Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1[END_REF][START_REF] Navab | Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3[END_REF] and also has an anti-inflammatory effect both by inhibiting secretion of interleukin-1␤ (IL-1␤) and tumour necrosis factor-␣ (TNF-␣) [START_REF] Hyka | Apolipoprotein A-I inhibits the production of interleukin-1beta and tumor necrosis factor-alpha by blocking contact-mediated activation of monocytes by T lymphocytes[END_REF] and via the removal of reactive oxygen species. It has been suggested that ABCA1 may be linked to these functions via apoA1. [START_REF] Reddy | ATP-binding cassette transporter 1 participates in LDL oxidation by artery wall cells[END_REF] The apoA1 concentration in the extracellular fluid is dependent on synthesis, catabolism, dissociation and its reassociation with the lipoproteins in the plasma compartment represented by the HDL component. [START_REF] Aiello | ABCA1-deficient mice: insights into the role of monocyte lipid efflux in HDL formation and inflammation[END_REF] This in turn could potentially influence lipid efflux in the plaque environment.

ABCA1 and the atherosclerotic plaque

The atherosclerotic plaque is a dynamic structure composed of lipids, cells and extracellular matrix. Plaques with high levels of collagen and smooth muscle cells providing a thick fibrous cap are characteristic of an advanced stable plaque, while unstable plaques have a thin cap, dense inflammatory infiltrate and a large lipid core. [START_REF] Virmani | Lessons from sudden coronary death: A comprehensive morphological classification scheme for atherosclerotic lesions[END_REF] Unstable plaques are prone to rupture with subsequent in situ thrombosis and embolism, [START_REF] Ross | Mechanisms of disease: atherosclerosis -an inflammatory disease[END_REF] leading to significant clinical consequences. The behaviour of these plaques is highly unpredictable and the precise mechanisms causing the transition to instability are incompletely understood. The development and subsequent progression of plaques are determined by changes in its three major constituents, and ABCA1 could potentially influence all of the processes governing plaque architecture, namely:

(1) lipid accumulation and metabolism;

(2) alterations in cell turnover involving apoptosis and inflammation; (3) altered extracellular matrix turnover.

ABCA1 and lipid metabolism

A large lipid core in the atheromatous plaque is one of the major indicators of potential instability. Inefficient macrophage function to clear lipids and cell debris could lead to an increase in the volume of lipid within the central core of the plaque. In the Tromso study [START_REF] Mathiesen | Low levels of highdensity lipoprotein cholesterol are associated with echolucent carotid artery plaques: The Tromso study[END_REF][START_REF] Mathiesen | Echolucent plaques are associated with high risk of ischemic cerebrovascular events in carotid stenosis: The Tromso study[END_REF] it was reported that lipid rich echolucent carotid plaques are associated with low levels of HDL cholesterol and increased risk of ischemic cerebrovascular events independent of the degree of stenosis and cardiovascular risk factors. Furthermore low HDL levels were shown to be associated with increased carotid intima-media thickness independent of other risk factors. [START_REF] Alagona | Low HDL cholesterol concentration is associated with increased intima-media thickness independent of arterial stiffness in healthy subjects from families with low HDL cholesterol[END_REF] Cholesterol from peripheral cells is returned to the liver by ABCA1 dependent and independent mechanisms. ApoA1 that is secreted or regenerated from HDL by the liver is lipidated by ABCA1 in peripheral cells to form pre-␤ HDL (nascent HDL) which then collects cholesterol from various cells. Although a basal level of phospholipidation of apoA1 may occur through an ABCA1 independent pathway, [START_REF] Kiss | The lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways[END_REF] the major part of it is done by ABCA1. It is also thought that the cholesterol processed from LDL receptor uptake in the liver is transferred to HDL by hepatic ABCA1 to be secreted into bile. In addition ABCA1 might also be involved in the removal of FC through apoA1 independent mechanisms via the formation of large membrane FC rich vesicles. [START_REF] Liu | Effects of apolipoprotein A-I on ATP-binding cassette transporter A1-mediated efflux of macrophage phospholipid and cholesterol: formation of nascent high density lipoprotein particles[END_REF] Animal studies have provided further proof of ABCA1 function. Classical examples are ABCA1 knock out [START_REF] Aiello | ABCA1-deficient mice: insights into the role of monocyte lipid efflux in HDL formation and inflammation[END_REF] and transgenic mice [START_REF] Cavelier | Regulation and activity of the human ABCA1 gene in transgenic mice[END_REF][START_REF] Singaraja | Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and ApoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1[END_REF][START_REF] Singaraja | Increased ABCA1 activity protects against atherosclerosis[END_REF][START_REF] Vaisman | ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice[END_REF] as well as the Wisconsin Hypoalpha Mutant Chicken (WHAM) [START_REF] Poernama | Spontaneous high density lipoprotein deficiency syndrome associated with a Z-linked mutation in chickens[END_REF][START_REF] Schreyer | Hypercatabolism of lipoprotein-free apolipoprotein A-I in HDL-deficient mutant chickens[END_REF] which is the naturally occurring animal model of TD. In the context of human studies on ABCA1, a 2-3fold increase in cardiovascular disease in TD patients and obligate ABCA1 heterozygotes as compared to age and sex matched controls from the Framingham study [START_REF] Schaefer | Coronary heart disease prevalence and other clinical features in familial high-density lipoprotein deficiency (Tangier disease)[END_REF] was found. A recent study revealed that heterozygotes with ABCA1 mutations had lower amounts of cholesterol efflux, lower HDL concentrations and greater carotid intima-media thickness than the control group. [START_REF] Van Dam | Association between increased arterial-wall thickness and impairment in ABCA1driven cholesterol efflux: an observational study[END_REF] This study also indicated that the upper limit of normal intima-media thickness (0.80 mm) is reached in ABCA1 heterozygotes at the age of 55 as compared to 80 years in the control group.

In this context, a potentially variable role of ABCA1 in hepatocytes and macrophages towards its contribution to HDL formation has been suggested. Studies in mice have highlighted the importance of both hepatic and macrophage ABCA1. Mice specifically overexpressing ABCA1 [START_REF] Aiello | Increased atherosclerosis in hyperlipidemic mice with inactivation of ABCA1 in macrophages[END_REF] in hepatocytes and macrophages showed significant increases in HDL levels. However, selective inactivation studies of ABCA1 in macrophages showed clear evidence of increased atherosclerosis in hyperlipidemic mice [START_REF] Aiello | Increased atherosclerosis in hyperlipidemic mice with inactivation of ABCA1 in macrophages[END_REF] but had very minimal impact on HDL formation. [START_REF] Haghpassand | Monocyte/macrophage expression of ABCA1 has minimal contribution to plasma HDL levels[END_REF] Similarly mice selectively deficient in leukocyte ABCA1 developed large advanced atherosclerotic lesions without any change in plasma HDL levels. [START_REF] Van Eck | Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues[END_REF] This implies that hepatic ABCA1 may be responsible for the main contribution of ABCA1 to plasma HDL. Recent studies by Sahoo et al [START_REF] Sahoo | ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from murine hepatocytes[END_REF] seem to support this finding. Taken together, these findings suggest that hepatic ABCA1 exerts a generalized anti-atherogenic effect via its contribution to HDL formation while macrophage ABCA1 provides a peripheral anti-atherogenic effect on the vasculature.

However, interesting questions did arise by studying TD and ABCA1 deficient mice. First, the accumulation of cholesteryl esters in these conditions is localized primarily to macrophages and certain tissues. [START_REF] Oram | Tangier disease and ABCA1[END_REF] The reason for this observation is still under investigation, but it has been suggested that lipids accumulate predominantly in tissues with high cell turnover and a large population of macrophages. Secondly, TD patients have only a 4-fold increase in atherosclerotic disease compared to the general population, despite the fact that more severe atherosclerosis would be expected as a consequence of the complete absence of HDL.

It has been proposed [START_REF] Oram | Tangier disease and ABCA1[END_REF] that the source of cholesterol may differ between different macrophage subtypes with arterial macrophages primarily obtaining cholesterol from lipoproteins while other peripheral macrophages accumulate cholesterol from phagocytosed cell membranes. Thus despite ABCA1 deficiency and low HDL levels in TD patients, the expected aggressive atherosclerosis fails to develop in the background of a less atherogenic lipid profile (less than 40% LDL) which provides a potentially diminished cholesterol pool for arterial macrophages. Hence it may be speculated that in the presence of normal or increased levels of LDL, ABCA1 deficiency could potentially enhance atherosclerosis.

ABCA1 and alterations in cell turnover

Apoptosis

Apoptosis is known to play a major role in regulating cell turnover in the plaque. Apoptosis (Greek: leaffall), often used synonymously with programmed cell death, is a physiologically highly selective method to eliminate old or injured cells before the leakage of intracellular contents can induce an immune response. The initial phase of apoptosis has to be followed by an efficient phagocytic phase to render the process immunologically silent. Therefore recognition of an apoptotic cell by the phagocyte is crucial. After stimulation, PS, an anionic phospholipid which resides in the inner leaflet, is translocated to the outer leaflet, a process which is sufficient to guarantee recognition by the phagocyte. [START_REF] Fadok | Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages[END_REF] ABCA1 was found to be involved in PS translocation and in promoting engulfment of apoptotic cells. [START_REF] Hamon | ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine[END_REF] This study revealed that phagocytosis is impaired in ABCA1 deficient macrophages and that forced expression of ABCA1 can confer engulfment ability to non-phagocytic cells. ABCA1 null animals were found to manifest reduced externalization of PS and similar findings were obtained in other studies. [START_REF] Smith | Evaluation of the role of phosphatidylserine translocase activity in ABCA1mediated lipid efflux[END_REF] The externalization of PS is paramount in the atherosclerotic plaque due to its known tendency to promote procoagulant potential [START_REF] Mallat | Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: a role for apoptosis in plaque thrombogenicity[END_REF][START_REF] Wang | Thrombogenic role of cells undergoing apoptosis[END_REF] and the coagulation cascade. ABCA1 was also shown to interact with the Fas associated death domain (FADD) protein, [START_REF] Buechler | Molecular and functional interaction of the ATPbinding cassette transporter A1 with Fas-associated death domain protein[END_REF] a process which may play a role in regulating apoptosis.

Inflammation

Cellular cholesterol levels determine the levels of intracellular oxygenated sterols which in turn regulates LDL oxidation, activation of IL-1 [START_REF] Rosklint | Oxysterols induce interleukin-1[beta] production in human macrophages[END_REF] and promotion of apoptotic pathways. [START_REF] Napoli | Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells[END_REF] ABCA1 which is proven to selectively remove cytotoxic FC from the cell [START_REF] Kellner-Weibel | Cytotoxic cellular cholesterol is selectively removed by apoA-I via ABCA1[END_REF] could potentially control intracellular oxysterol levels. Macrophages and activated T lymphocytes in the plaque [START_REF] Schaub | Fas/FADD-mediated activation of a specific program of inflammatory gene expression in vascular smooth muscle cells[END_REF] release proinflammatory cytokines, mainly interferon-gamma (IFN-␥) from T lymphocytes), TNF and IL-1 (both from macrophages). These cytokines have been reported to decrease the mRNA and protein levels of ABCA1. [START_REF] Wang | Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1[END_REF][START_REF] Baranova | Lipopolysaccharide down regulates both scavenger receptor B1 and ATP binding cassette transporter A1 in RAW cells[END_REF][START_REF] Panousis | Interferon-gamma induces downregulation of Tangier disease gene (ATP-bindingcassette transporter 1) in macrophage-derived foam cells[END_REF][START_REF] Ruan | PPAR agonists protect mesangial cells from interleukin 1[beta]-induced intracellular lipid accumulation by activating the ABCA1 cholesterol efflux pathway[END_REF][START_REF] Wang | Interferon-gamma-mediated downregulation of cholesterol efflux and ABC1 expression is by the Stat1 pathway[END_REF] Moreover, lipopolysaccharide found in infectious agents like Chlamydia was also reported to downregulate ABCA1 expression. [START_REF] Baranova | Lipopolysaccharide down regulates both scavenger receptor B1 and ATP binding cassette transporter A1 in RAW cells[END_REF][START_REF] Khovidhunkit | Endotoxin down-regulates ABCG5 and ABCG8 in mouse liver and ABCA1 and ABCG1 in J774 murine macrophages: differential role of LXR[END_REF] It has been demonstrated that IFN-␥ can cause a 3-fold downregulation of ABCA1 and simultaneously an increase in acyl-CoA: cholesterol acyltransferase (ACAT) activity [START_REF] Panousis | Interferon-gamma induces downregulation of Tangier disease gene (ATP-bindingcassette transporter 1) in macrophage-derived foam cells[END_REF][START_REF] Wang | Interferon-gamma-mediated downregulation of cholesterol efflux and ABC1 expression is by the Stat1 pathway[END_REF] thus promoting atherosclerosis. IL-1␤, responsible for the activation of MMPs and other interleukins have also been reported to inhibit ABCA1 function thus promoting lipid accumulation. [START_REF] Ruan | PPAR agonists protect mesangial cells from interleukin 1[beta]-induced intracellular lipid accumulation by activating the ABCA1 cholesterol efflux pathway[END_REF] It has been recently reported that myeloperoxidase present in atheromas [START_REF] Daugherty | Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions[END_REF] promotes atherogenesis by impairing ABCA1 function. [START_REF] Bergt | The myeloperoxidase product hypochlorous acid oxidizes HDL in the human artery wall and impairs ABCA1-dependent cholesterol transport[END_REF] There is evidence that ABCA1 is involved in the secretion of IL-1␤ and macrophage inhibitory factor (MIF) from macrophages and monocytes respectively. [START_REF] Hamon | Interleukin-1beta secretion is impaired by inhibitors of the ATP binding cassette transporter, ABC1[END_REF][START_REF] Zhou | The ATP binding cassette transporter A1 contributes to the secretion of interleukin 1beta from macrophages but not from monocytes[END_REF][START_REF] Flieger | Regulated secretion of macrophage migration inhibitory factor is mediated by a non-classical pathway involving an ABC transporter[END_REF] However, the role of ABCA1 in the transport of these proinflammatory cytokines needs further evaluation.

Hence it could be speculated that a vicious cycle might ensue where the initial cytokine induction could cause ABCA1 deficiency, which in turn results in inadequate clearance of apoptotic and necrotic cells that further aggravates inflammatory mediators leading to plaque instability. All of the previously mentioned findings suggest that therapeutic upregulation of ABCA1 expression could be a promising approach in the context of treatment of atherosclerosis.

ABCA1 and extracellular matrix turnover

The integrity of the extracellular matrix (ECM) which influences the strength of the fibrous cap in the plaque [START_REF] Tunon | Regulation of matrix proteins and impact on vascular structure[END_REF] is determined by the balance between matrix metalloproteinases (MMP) and tissue inhibitors of MMPs. [START_REF] Faia | Matrix metalloproteinases and tissue inhibitors of metalloproteinases in hamster aortic atherosclerosis: Correlation with in-situ zymography[END_REF] There is general agreement that increased levels of intra-plaque MMPs could lead to plaque rupture [START_REF] Libby | Macrophages and atherosclerotic plaque stability[END_REF] and clinically significant plaque instability has been shown to be associated with increased levels of MMP-9. [START_REF] Loftus | Increased matrix metalloproteinase-9 activity in unstable carotid plaques. A potential role in acute plaque disruption[END_REF] The MMPs which are secreted by inflammatory cells promote VSMC migration and ECM degradation. [START_REF] Galis | Matrix metalloproteinases in vascular remodeling and atherogenesis: The good, the bad, and the ugly[END_REF] The proinflammatory cytokine IL-1␤ is reported to upregulate MMP-9 production. [START_REF] Amin | Secretion of matrix metalloproteinase-9 by the proinflammatory cytokine, IL-1beta: A role for the dual signalling pathways, Akt and Erk[END_REF][START_REF] Yoo | IL-1beta induces MMP-9 via reactive oxygen species and NF-kappaB in murine macrophage RAW 264.7 cells[END_REF] The interaction of ABCA1 with apoA1 may reduce MMP-9 production as apoA1 is documented to suppress cytokine IL-1␤ production. [START_REF] Hyka | Apolipoprotein A-I inhibits the production of interleukin-1beta and tumor necrosis factor-alpha by blocking contact-mediated activation of monocytes by T lymphocytes[END_REF] Furthermore the nuclear receptors liver-X-receptor (LXR) and peroxisome proliferator-activated receptor (PPAR), which are the key regulators of ABCA1 function, have documented anti-inflammatory and anti-MMP activity. [START_REF] Castrillo | Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages[END_REF][START_REF] Jiang | PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines[END_REF][START_REF] Joseph | Reciprocal regulation of inflammation and lipid metabolism by liver X receptors[END_REF][START_REF] Li | Peroxisome proliferator-activated receptor gamma-dependent repression of the inducible nitric oxide synthase gene[END_REF][START_REF] Marx | Macrophages in human atheroma contain PPARgamma: Differentiation-dependent peroxisomal proliferator-activated receptor gamma (PPARgamma) expression and reduction of MMP-9 activity through PPARgamma activation in mononuclear phagocytes in vitro[END_REF]

Regulation of ABCA1

The pivotal role played by ABCA1 in cholesterol homeostasis mandates a tight regulatory pathway. In addition to increased lipid efflux, a consequence of ABCA1 overexpression could also be the potential alteration of membrane structure with subsequent detrimental effects. [START_REF] Wang | Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1[END_REF] Evidence for the tight regulation of ABCA1 is the short half life of the ABCA1 protein [START_REF] Arakawa | Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting it from thiol protease-mediated degradation[END_REF][START_REF] Oram | ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages[END_REF] and its rapid turnover in macrophage cell lines. [START_REF] Wang | Unsaturated fatty acids inhibit cholesterol efflux from macrophages by increasing degradation of ATPbinding cassette transporter A1[END_REF] It is regulated at transcriptional and posttranscriptional levels (Figure 2).

Transcriptional regulation

Sterols

The large intracellular sterol concentration in foam cells plays a major role in ABCA1 regulation. Acetylated LDL upregulates ABCA1 mRNA and protein which can be reversed by incubation of these macrophages in HDL. [START_REF] Langmann | Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages[END_REF] It was also reported that ABCA1 mRNA and protein were upregulated in a time and dose dependent fashion by native LDL. [START_REF] Liao | Native LDL upregulation of ATP-binding cassette transporter-1 in human vascular endothelial cells[END_REF]

Nuclear receptors

Nuclear receptors are ligand activated transcription factors that regulate the expression of their target genes. The tissue specific expression and ligand availability tightly controls its activity. [START_REF] Chawla | nuclear receptors and lipid physiology: Opening the X-Files[END_REF] LXR and PPAR, belonging to a subgroup called adopted orphan nuclear receptor group, are the major players in ABCA1 regulation and will therefore be discussed below.

LXRs act as cholesterol sensors that respond to elevated sterol concentrations and activate a cadre of genes that govern transport, catabolism and elimination of cholesterol. [START_REF] Lu | Orphan nuclear receptors as eLiXiRs and FiXeRs of sterol metabolism[END_REF] LXR␣ expressed in the macrophage is important in terms of plaque physiology as it seems to be capable of activating and suppressing the ABCA1 gene based on ligand availability. [START_REF] Wagner | Promoter-specific roles for liver X receptor/corepressor complexes in the regulation of ABCA1 and SREBP1 gene expression[END_REF] The ligands that activate these receptors in the macrophage are mainly oxysterols like 27-hydroxycholesterol. [START_REF] Fu | 27-hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells[END_REF] LXR␣ influences ABCA1 expression transcriptionally and post-transcriptionally (Figure 3). Studies have reported that the oxysterol induction of ABCA1 is partly through the LXR pathway. [START_REF] Repa | Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers[END_REF][START_REF] Venkateswaran | Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha[END_REF] Recent studies have highlighted the anti-atherogenic activity of LXR. 100,101 In human macrophages exclusively, it has been observed that the LXR␣ gene itself is a target for the LXR signaling pathway, an effective autoregulatory mechanism to amplify the ABCA1 lipid efflux pathway. 102,103 PPARs have been implicated in the regulation of both inflammation and lipid homeostasis in the macrophage and polyunsaturated fatty acids serve as their ligands. [START_REF] Chawla | nuclear receptors and lipid physiology: Opening the X-Files[END_REF] PPAR␥ enhances cholesterol efflux by inducing the transcription of LXR␣ and thus ABCA1. 104,105 The observation that PPAR agonists and LXR ligands are ineffective in macrophages of TD patients [START_REF] Venkateswaran | Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha[END_REF]105 indicates that functional ABCA1 is required for this pathway of cholesterol efflux. With regard to the role of these nuclear receptors in the context of plaque pathophysiology, LXR␣ and PPAR␥ have been documented to have anti-inflammatory [START_REF] Jiang | PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines[END_REF][START_REF] Joseph | Reciprocal regulation of inflammation and lipid metabolism by liver X receptors[END_REF][START_REF] Li | Peroxisome proliferator-activated receptor gamma-dependent repression of the inducible nitric oxide synthase gene[END_REF] and anti-MMP activity. [START_REF] Castrillo | Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages[END_REF][START_REF] Marx | Macrophages in human atheroma contain PPARgamma: Differentiation-dependent peroxisomal proliferator-activated receptor gamma (PPARgamma) expression and reduction of MMP-9 activity through PPARgamma activation in mononuclear phagocytes in vitro[END_REF] The PPAR␥-LXR␣ -ABCA1 cascade does represent a powerful means of cholesterol efflux from the macrophage and therefore could play a significant role in influencing the progression of atherosclerotic plaques.

Post-transcriptional regulation

Studies in mice have revealed a significant discordance between ABCA1 protein and mRNA levels, suggesting that post-transcriptional regulation plays a major role [START_REF] Wellington | ABCA1 mRNA and protein distribution patterns predict multiple different roles and levels of regulation[END_REF] in ABCA1 protein expression. Unsaturated fatty acids have been found to promote ABCA1 protein degradation directly [START_REF] Wang | Unsaturated fatty acids inhibit cholesterol efflux from macrophages by increasing degradation of ATPbinding cassette transporter A1[END_REF]106 and indirectly. 107,108 This may be significant in disorders like type 2 diabetes and insulin resistance, conditions with increased levels of fatty acids where accelerated atherosclerosis is observed. Cytotoxic levels of intracellular FC 109 were also found to promote ABCA1 protein degradation through the proteolysis pathway. ABCA1 phosphorylation, which is reported to be influenced by apoA1 110 and protein kinase C, 111 may also have a major effect on protein stability. The PEST sequence identified in ABCA1 by Wang et al 112 appears significant in protein degradation as deletion of this motif resulted in a 4-5-fold increase in ABCA1 protein, increased ABCA1 mediated efflux and enhanced apoA1 binding.

In vitro experiments with peritoneal macrophages, transfected cells and mouse primary hepatocytes have shown that apoA1 binding increased ABCA1 protein without affecting mRNA levels. 112 Interestingly this apoA1 mediated stabilization of ABCA1 protein is achieved by inhibition of PEST sequence mediated degradation by proteases. [START_REF] Arakawa | Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting it from thiol protease-mediated degradation[END_REF]112 There is also evidence that PLTP interacts with ABCA1 for its function in cholesterol efflux and also stabilizes ABCA1 protein. 113 We have reported that, in human carotid atherosclerotic plaques, ABCA1 protein is significantly reduced despite increased mRNA. 114 The observed upregulation in this study of both LXR␣ and ABCA1 mRNA in atherosclerotic tissues could be attributed to the oxysterol-rich environment inside the plaque potentially amplified by low ABCA1 protein levels. It is possible that the degradation of ABCA1 protein in the plaque microenvironment might be the key factor influencing cholesterol homeostasis at the macrophage level. The resulting localized deficiency in ABCA1 function could lead to decreased lipid efflux, accumulation of oxysterols and acceleration of the atherosclerotic process.

Therapeutic modulation

The nuclear receptors PPAR␥ and LXR␣ are promising targets for pharmacological manipulation of the ABCA1 pathway. In vitro studies have shown that PPAR agonists such as the glitazones used in type 2 diabetes induce cholesterol efflux from macrophages through the activation of ABCA1. 105,115 It is interesting to note that PPAR agonists can effectively override the cytokine IL-1␤ induced suppression of ABCA1 and promote cholesterol efflux. [START_REF] Ruan | PPAR agonists protect mesangial cells from interleukin 1[beta]-induced intracellular lipid accumulation by activating the ABCA1 cholesterol efflux pathway[END_REF] This effect in itself may be valuable in terms of plaque pathophysiology. ACAT inhibitors which increase intracellular FC levels have also been reported to upregulate ABCA1 both at the mRNA and protein level 116 but are unlikely to be considered for general use.

Studies on rats showed that clofibrate, widely used as a hypolipidemic agent, increases ABCA1 mRNA through activation of LXR␣. 117 Recently verapamil was shown to enhance both ABCA1 mRNA and protein expression through LXR independent mechanisms. 118 Ando et al 119 demonstrated that in vivo pravastatin increased LXR␣ mRNA levels. Statins increase HDL levels in addition to the significant reductions in total cholesterol and triglycerides. 120,121 In fact it is suggested that the increase in HDL cholesterol produced by statins could partly also be through the upregulation of ABCA1 and apoA1. 122,123 PPAR and LXR agonism may be beneficial in terms of its generalized effect on HDL and inflammation but its effect on the localized atheromatous plaque has not been studied in detail.

The benefits of transcriptional upregulation may be potentially annulled by the in vivo plaque microenvironment that promotes ABCA1 protein degradation. 114 In addition to ABCA1 transcriptional upregulation, ABCA1 protein stabilization may be crucial to promote function of cholesterol efflux.

ABCA1 and atherosclerosis 115

Figure 3 Influence of nuclear receptors LXR␣ and PPAR␥ on ABCA1 expression. LXR␣ has been shown to activate the sterol regulatory element binding protein-1 (SREBP-1) 124 and stearoyl-CoA desaturases-1 and 2 (SCD) 108 which results in increased unsaturated fatty acid synthesis. These unsaturated fatty acids serve as ligands for PPAR activation [START_REF] Chawla | nuclear receptors and lipid physiology: Opening the X-Files[END_REF] but have been implicated in causing ABCA1 protein degradation. [START_REF] Wang | Unsaturated fatty acids inhibit cholesterol efflux from macrophages by increasing degradation of ATPbinding cassette transporter A1[END_REF]106 Therefore the enhanced ABCA1 transcription induced by LXR ligands may be counteracted by increased ABCA1 protein degradation.

Synthetic peptides based on the structure of apoA1 have been found to be effective in stabilizing ABCA1 protein. 125,126 Calpain protease inhibitors and specific LXR agonists 127 are further venues for exploration in the quest to promote cholesterol efflux therapeutically. Therefore it would be interesting to assess the effect of combined transcriptional upregulation with protein stabilization of ABCA1 in the context of treatment of atherosclerosis.

Conclusion

The critical role of ABCA1 at the macrophage and hepatic level regarding lipid efflux and HDL formation could have a major influence on the biogenesis and progression of atheromatous plaques. Further studies need to be focused on apoA1 and ABCA1 interactions that seem to regulate each other and have a potent role in protection against atherosclerosis. The PPAR-LXR-ABCA1 cascade has a substantial role in cholesterol homeostasis and inflammation and therefore has a promising potential for therapeutic manipulation. The efficacy of combined therapy with ABCA1 transcriptional upregulation and protein stabilization needs to be evaluated. Further work needs to be undertaken to unravel other functions of this transporter and its pathways so that therapeutic strategies could be devised to prevent atherosclerotic disease and its complications.

Figure 1

 1 Figure 1The influence of ABCA1 on the atherosclerotic plaque.

Figure 2

 2 Figure 2 Regulation of ABCA1 at transcriptional and post-transcriptional levels.

Vascular Medicine 2005; 10: 109-119

Acknowledgements

The Stroke Association and the Medical Research Council support our work on ABCA1.