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54 Bernardo Trigatti

pathways appear to be involved. For example, HDL is a carrier of antioxidant vitamins (such as α-tocopherol—vitamin E), which help protect cells from harmful effects of reactive oxygen species [4, 5]. HDL is also associated with enzymes such as paraoxonase (PON) and platelet activating factor acylhydrolase (PAF-AH) that may play important roles in the removal of oxidized lipids from other lipoproteins such as low-density lipoprotein (LDL) [6–8]. HDLs also trigger signaling events within cells leading to diverse consequences, including the activation of endothelial nitric oxide synthase (eNOS) and protection of cells in the vascular wall against apoptosis, processes which have been associated with protection against atherosclerosis [9–18].

HDL also plays a key role in reverse cholesterol transport. This is the removal of cholesterol from cells in peripheral tissues (e.g., macrophages and foam cells in the artery wall), and its delivery to the liver. HDL-associated cholesterol is converted to cholesteryl ester by lecithin cholesterol acyltransferase (LCAT) [19, 20]. HDL-associated cholesteryl ester can be transferred to very low-density lipoprotein (VLDL) in exchange for triglycerides by cholesteryl ester transfer protein (CETP) [21]. Alternatively, cholesteryl ester that remains associated with HDL can be taken up by liver hepatocytes via a pathway called selective lipid uptake. This involves the net internalization of only the lipid portion of the HDL particle, without the net internalization and degradation of the HDL particle itself (reviewed in [22]). Internalized cholesterol can be secreted into bile either as cholesterol or after conversion to bile acids or be repackaged into nascent VLDL and secreted from the hepatocyte into the circulation (reviewed in [22]). HDL has also been implicated in the reverse transport of oxidized lipids, derived from cells or from other lipoproteins, to the liver for clearance [23]. Therefore, the “reverse cholesterol transport” pathway may play important roles in the clearance of diverse lipids in addition to cholesterol, highlighting the importance of this pathway to protection against atherosclerosis.

The Scavenger Receptor Class B Type I

Almost a decade ago, Krieger and coworkers [24] identified the scavenger receptor class B type I (SR-BI) as an HDL receptor that could mediate the selective uptake of HDL lipids. SR-BI is a member of the CD36 family of proteins (see Chapter 5) and contains two transmembrane domains, short N-terminal and C-terminal cytoplasmic regions, and a large extracellular region that is heavily glycosylated (reviewed in [22], see Fig. 4.1). SR-BI is also palmitoylated on two Cys residues near the junction between the C-terminal cytoplasmic and transmembrane regions [25, 26]. It is expressed most abundantly in cells with a high capacity for selective HDL lipid uptake—steroidogenic cells and hepatocytes (reviewed in [22]). This suggested that SR-BI may play an important role in the later stages of reverse

SR-BI

c c

N

c

FIGURE 4.1. SR-BI and known ligands. SR-BI is represented with two transmembrane regions residing in the membrane (gray bar), cytoplasmic C-terminus and N-terminus, and a large extracellular region. Approximate locations of N-linked glycosylation sites are shown in gray, as are the locations of two cysteine residues that are sites of palmitoylation. Known SR-BI ligands are listed at the top and include lipoproteins, chemically modified proteins, and lipoproteins including advance glycation endproduct modified BSA (AGE-BSA), liposomes containing anionic phospholipids (PL), apoptotic cells, bacterial lipopolysaccharide, and the acute phase protein serum amyloid alpha (SAA) (either free or associated with lipoproteins) (reviewed in [27] and [86]).

cholesterol transport and transport of HDL cholesterol to steroidogenic tissues. SR-BI is also expressed in a variety of other cell types (reviewed in [22, 27, 28]) including macrophages and endothelial cells [29–32] and could therefore influence atherosclerosis locally at the vessel wall (explained later).

When overexpressed in transfected cells, SR-BI can mediate both increased selective uptake of HDL lipids and increased efflux of free cholesterol tracer to HDL acceptors [24, 33, 34]. This has led to the notion that SR-BI can mediate the bidirectional flux of lipids between cells and bound lipoproteins and the suggestion that the direction of net flux is dependent on the concentration gradient of the lipid [35]. For example, in Chinese hamster ovaryderived cells, which have relatively low levels of intracellular cholesterol, the net flux appears to be in the direction of the cell [24]. The observation that SR-BI can mediate the efflux of unesterified cholesterol tracer from cells has led to the suggestion that it may play an important role in HDL-dependent

56 Bernardo Trigatti

cholesterol efflux from macrophages (see [33, 34]). Consequently, SR-BI has been implicated in both early steps (i.e., cholesterol efflux from macrophages) and late steps (hepatic selective HDL cholesterol uptake) in reverse cholesterol transport.

SR-BI has also been implicated in mediating HDL-dependent signaling events in endothelial cells (see Chapter 5). For example, the HDL-dependent stimulation of eNOS appears to be dependent on SR-BI [9, 11, 12, 14]. This may occur via the transfer of bioactive lipids (estradiol, ceramide, and/or sphingosine-1-phosphate) with signaling activity and/or through the generation of intracellular signaling events including the activation PKB/Akt pathways [9, 11, 12, 14, 17]. The ability of SR-BI to mediate HDL-dependent eNOS activation may also be related to SR-BI-mediated cholesterol efflux to HDL [36]. The role of SR-BI in cholesterol efflux from cells appears to be controversial, however. Overexpression of SR-BI in transfected cells has been shown quite clearly to increase the efflux of cholesterol tracer to HDL [33, 34, 37]. In contrast, several studies have indicated that SR-BI is not required for cholesterol efflux to HDL in different cells including endothelial cells [38] and macrophages [39–41].

Certain tissues, including the liver, contain an alternatively spliced transcript giving rise to an isoform called SR-BII, which differs from SR-BI in its C-terminal cytoplasmic region [42, 43]. SR-BI appears to be considerably more active than SR-BII in mediating lipid transfer between cells and lipoproteins [44]. This may be the result of a different subcellular distribution of the two isoforms, with SR-BI demonstrating a greater distribution on the plasma membrane than does SR-BII [44].

Recently, Smart and coworkers [18] have reported that SR-BI expression induces apoptosis in cells in the absence of ligand stimulation. Both HDL interaction with SR-BI and eNOS activation were able to inhibit SR-BI-triggered apoptosis. This suggests the possibility that SR-BI could contribute to apoptosis of endothelial cells or other cells in an HDLand eNOS-regulated manner. This process could contribute to endothelial dysfunction and initiation or development of atherosclerosis, and HDL and eNOS might act, at least in part, by halting this process [18].

In addition to mediating the binding of HDL and HDL-dependent lipid exchange with cells, SR-BI also binds a variety of other ligands (Fig. 4.1). These include other lipoproteins (LDL, VLDL, and chylomicrons), modified proteins/lipoproteins (e.g., advance glycation end-product modified bovine serum albumin (AGE-BSA), oxidized or acetylated LDL), the acute phase protein serum amyloid alpha (SAA—either free or bound to HDL), phospholipid (PL) vesicles containing anionic PL, apoptotic cells, and bacterial cell surface components (e.g., lipopolysaccharide) (see [27] for recent review). Therefore in addition to playing a role in HDL metabolism, SR-BI may play roles in a variety of other physiological processes, many of which may affect the development or progression of atherosclerosis.

SR-BI and Atherosclerosis: Studies of Gene-Targeted

Mice

Studies of genetically manipulated mice have provided substantial insight into SR-BI’s physiological role in HDL metabolism and atherosclerosis. SR-BI knockout (KO) mice, in which the gene encoding SR-BI (Scarb1) has been inactivated by targeted mutagenesis, exhibit no detectable SR-BI expression in tissues examined [45]. These mice have increased cholesterol levels associated with enlarged HDL particles with altered compositions [45–48]. The enlarged HDL particles contain increased apoE and nonesterified/esterified cholesterol ratios accompanied by decreased levels of LCAT activity [45, 47–49]. These alterations in plasma HDL composition and structure are accompanied by reduced lipid storage in steroidogenic tissues and reduced biliary cholesterol secretion [45, 46, 50]. Mice that are heterozygous for the targeted mutation express half of normal SR-BI levels in tissues that have been analyzed, including liver and adrenal glands [45]. These mice have slightly increased HDL cholesterol, and ~50% reductions in adrenal cholesterol levels [45]. SR-BI KO mice do not, however, exhibit altered hepatic cholesterol levels or cholesterol biosynthesis [49, 50]. SR-BI KO mice have been reported to exhibit increased levels of hepatic expression of ABCG1, an ATP-binding cassette transporter (ABC) implicated in cholesterol efflux to HDL, and mice carrying one or two copies of the inactivating mutation exhibit reduced hepatic ABCG5 and ABCG8, other ABC family members which have been implicated in regulating hepatic cholesterol secretion into bile [49, 51, 52].

SR-BI KO mice have reduced hepatic and adrenal uptake of HDL cholesterol and exhibit reduced selective clearance of HDL and LDL cholesterol from plasma [53–55]. These studies suggest that SR-BI is required for selective uptake of cholesterol from HDL and LDL by liver and steroidogenic cells in vivo [45, 46, 50, 53–56]. SR-BI KO mice have also been reported to exhibit a greater increase in plasma triglycerides after an intragastric fat load whereas adenovirus-infected mice with hepatic overexpression of SR-BI show a reduced response, relative to controls [57, 58]. This combined with in vitro studies, demonstrating reduced binding of chylomicron-like particles to hepatocytes from SR-BI KO mice relative to controls, suggest that SR-BI may also be involved in chylomicron metabolism in vivo [57, 58].

SR-BI KO mice develop increased atherosclerosis either when challenged with high-fat/atherogenic diets or when SR-BI deficiency is combined with deficiency in genes encoding either LDL receptor or apoE [39, 46, 49]. Mice deficient in SR-BI alone develop increased diet-induced atherosclerosis compared to either heterozygous or homozygous SR-BI-expressing mice [49]. Examination of gene expression in aortas from these mice has revealed increased expression of genes involved in monocyte adhesion to endothelial cells, including P- and E-selectins and vascular cell adhesion molecule (VCAM)-1; similar increases were seen in CD68, apoE, ABCA1, and ABCG1,