Macrophage Cholesterol Efflux and Reverse Cholesterol Transport

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The uptake of modified LDLs in arterial macrophages leads to the cellular accumulation of cholesterol and oxidized lipids. This process causes the macrophages to transform into lipid-laden macrophage foam cells which can no longer escape the arterial wall (Potteaux et al. 2011). The excessive cellular cholesterol in macrophage foam cells can be efficiently removed by the potent cholesterol acceptors lipid-free apoA-I and HDLs to be transported back to the liver by a process termed reverse cholesterol transport.

4.3.1 Transendothelial HDL Transport

In order to reach these resident macrophage foam cells in the innermost layer of the arterial wall, apoA-I and HDLs first need to passage across the endothelial cell layer. There is cell culture evidence that lipid-free apoA-I binds to specific saturable high-affinity binding sites on aortic endothelial cells, which is followed by internalization, transcytosis, and delivery of lipidated apoA-I to the basolateral side (Rohrer et al. 2006). A similar phenomenon was observed when cultivated aortic endothelial cells were treated with mature HDLs (Rohrer et al. 2009). However, the mechanisms involved in regulating this endothelial transport of apoA-I and HDLs are not fully understood. The ABCA1 transporter is thought to play major role in the apical-to-basolateral transport of apoA-I in aortic endothelial cells (Cavelier et al. 2006). Induction of ABCA1 expression by a combination of oxysterol and 9-cis-retinoic acid increased the binding and internalization of apoA-I in endothelial cells, whereas knockdown of endothelial ABCA1 by RNA interference reduced the cellular uptake and transcytosis of apoA-I (Cavelier et al. 2006). Conversely, siRNA-mediated gene silencing experiments revealed that the cell surface binding and transport of HDLs through aortic endothelial cells are highly dependent on SR-BI and ABCG1, but not on ABCA1 (Rohrer et al. 2009). Subsequent research has demonstrated expression of the ectopic β-chain of F0F1 ATPase (β-ATPase) on the endothelial cell surface (Cavelier et al. 2012). The results of this study further suggest that the binding of lipid-free apoA-I to endothelial cell surface β-ATPase facilitates the uptake and transport of lipidated apoA-I and mature HDLs by enhancing β-ATPase-mediated hydrolysis of extracellular ATP into ADP and inducing consecutive activation of the purinergic P2Y12 receptor (Cavelier et al. 2012). More recently, transendothelial transport of HDL particles was found to be modulated by endothelial lipase both through its enzymatic lipolytic activity and its ability to bridge the binding of lipoproteins to the endothelial surface (Robert et al. 2013). Initiation of an atherosclerosis-related inflammatory phenotype after stimulation of vascular endothelial cells with interleukin-6 significantly increased the binding, cell association, and transport of HDLs, which was linked mechanistically to the increase in endothelial lipase expression in response to interleukin-6 (Robert et al. 2013).

4.3.2 Cholesterol Efflux from Macrophages

Once apoA-I and HDLs have reached macrophage foam cells in the atheromatous vessel wall, macrophage cholesterol efflux can be elicited via several different pathways. It is well documented that lipid-free apoA-I and pre-beta HDL particles are able to remove cholesterol and phospholipids from macrophage foam cells via the ABCA1 transporter (Wang et al. 2000; Wang and Tall 2003). It has been postulated that apoA-I-dependent cholesterol efflux from cells is mediated either by a direct protein–protein interaction between apoA-I and ABCA1 or indirectly by ABCA1-induced changes in the membrane cholesterol distribution (Wang and Tall 2003). The ability of ABCA1 to mobilize cellular lipids to apoA-I is essential for the initial lipidation of apoA-I. The ABCG1 transporter is responsible for a major part of the macrophage cholesterol efflux towards mature HDL particles (Wang et al. 2004). Since cellular removal of cholesterol via ABCG1 was not accompanied by specific binding of HDLs to the plasma membrane (Wang et al. 2004), the molecular basis of efflux of cholesterol mediated by ABCG1, like ABCA1, is not fully understood and warrants further investigation. Another cell surface receptor involved in macrophage cholesterol efflux elicited by mature HDLs is SR-BI. Binding of HDL to macrophage SR-BI facilitates a bidirectional exchange of unesterified cholesterol and other lipids between the cell membrane and HDL acceptor particles according to the cholesterol concentration gradient (de La Llera-Moya et al. 2001). This cholesterol concentration gradient between cells and HDL not only allows cholesterol transfer via SR-BI but also passive diffusion of cholesterol molecules to nearby HDL particles by receptor-independent processes (von Eckardstein et al. 2001; Yancey et al. 2003). The HDL-associated enzyme LCAT catalyzes the esterification of cholesterol in HDL particles (Calabresi and Franceschini 2010). The generated cholesteryl esters leave the particle surface and form the core of the maturing HDL particle; hence, a local gradient of unesterified cholesterol is created that maintains a continuous flow of unesterified cholesterol from macrophages towards HDLs (Calabresi and Franceschini 2010).

4.3.3 Exit from the Arterial Wall

There is little reported data on the route used by HDLs to leave the arterial intima. Recent lines of evidence support a role for the lymphatic vasculature in the transport of HDLs from the interstitial space back to the bloodstream. Following injection of fluorescently labeled HDLs in the mouse footpad, HDLs were subsequently found in the draining lymph node and its afferent and efferent lymphatic vessels (Lim et al. 2013). Moreover, the movement of cholesterol from macrophages in the footpad to the lymph and plasma was substantially reduced in mice with surgical interruption of the afferent lymphatic vessels or in Chy mutant mice that lack dermal lymphatic capillaries (Lim et al. 2013; Martel et al. 2013). The importance of the lymphatic system in the reentry of cholesterol originating from macrophages via HDLs in the circulation was further confirmed by additional experiments showing that the appearance of macrophage-derived cholesterol in the plasma was impaired after implantation of [3H]-cholesterol-labeled macrophages in the tail skin of apoA-I transgenic mice with microsurgical ablation of the major lymphatic conduits in the tail (Martel et al. 2013). To provide more direct proof for the involvement of the lymphatic vasculature in the transport of cholesterol from the atherosclerotic plaque, aortic segments with advanced atherosclerotic lesions were loaded with D6-cholesterol and transplanted in apoE knockout mice (Martel et al. 2013). Inhibition of regrowth of a functional lymphatic vasculature in the aortic donor wall by an antibody blocking the function of vascular endothelial growth factor receptor 3 markedly suppressed the apoE-induced removal of D6-cholesterol from the transplanted aorta (Martel et al. 2013), hence emphasizing the pivotal role of the lymphatic system for the egress of cholesterol from the aortic wall. The entry of HDL particles into lymphatic vessels appears to be primarily dependent on SR-BI. Experiments in cultured cells demonstrate that the internalization and transcytosis of HDL particles by lymphatic endothelial cells are diminished by an SR-BI blocking antibody or a selective SR-BI inhibitor (Lim et al. 2013). In agreement, lymphatic transport of HDLs is compromised in SRBI-null mice and in mice treated with an antibody against SR-BI (Lim et al. 2013). Although not formally tested, it is also possible that, in order to reach the circulation again, HDL particles undergo transcytosis through the luminal endothelial cell barrier or transfer cholesterol to endothelial cells for delivery to circulating HDLs. The importance of a well-regulated homeostasis between entry and exit of HDL particles into and from the arterial wall, respectively, is indicated by the enrichment of modified and dysfunctional HDLs and apoA-I in atherosclerotic lesions (DiDonato et al. 2013, 2014; Huang et al. 2014).

4.3.4 Delivery of Cholesterol to the Liver and Intestine

After having left the arterial wall and entered the circulation, the HDL particles transport the cholesterol either directly or indirectly via apoB-containing lipoproteins to the liver for either secretion into bile or for de novo assembly of lipoproteins. The first direct way by which HDL can deliver cholesterol to the liver is via SR-BI, a cell surface receptor that binds HDLs and mediates the selective uptake of HDL-associated cholesteryl esters and the subsequent resecretion of cholesteryl ester-poor HDLs (Acton et al. 1996). Secondly, HDL particles can be taken up from the circulation by the liver via HDL holoparticle endocytosis (i.e., uptake of the whole HDL particle). The binding of apoA-I to the ectopic β-chain of F0F1 ATPase on hepatocytes triggers extracellular production of ADP, which in turn acts on the P2Y13 receptor to stimulate HDL holoparticle endocytosis (Martinez et al. 2003; Jacquet et al. 2005). In humans, the major fraction of the cholesteryl esters in HDL particles are shuttled via the action of CETP to apoBcontaining lipoproteins (Charles and Kane 2012). These cholesteryl esters originating from HDLs are internalized as part of apoB-containing lipoproteins by hepatic LDL receptors. Following hepatic uptake, HDL-derived cholesterol is, at least in part, targeted for fecal excretion via the biliary route (Lewis and Rader 2005; Nijstad et al. 2011). Hepatic cholesterol can either be secreted directly in the free form into the bile by the ABCG5/G8 heterodimer or alternatively be converted by cholesterol 7α-hydroxylase (CYP7A1) and other enzymes into bile acids for hepatic excretion via the bile salt export pump (BSEP) (Dikkers and Tietge 2010). Secretion into bile enables cholesterol to move from the hepatocyte to the intestinal lumen for final excretion via the stool. This major HDL-driven pathway to eliminate atherogenic cholesterol from the body is also known as reverse cholesterol transport (Annema and Tietge 2012). Although reverse cholesterol transport from plaque macrophages to feces only represents a very small proportion of the total reverse cholesterol transport flux from peripheral tissues, it is key component of the reverse transport potentially capable of initiating and maintaining the regression of atherosclerosis (Lee-Rueckert et al. 2013).

 
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