Distinct mechanisms for OxLDL uptake and cellular trafficking by class B scavenger receptors CD36 and SR-BI

Modified forms of LDL, including oxidized low density lipoprotein (OxLDL), contribute to macrophage lipid accumulation in the vessel wall. Despite the pathophysiological importance of uptake pathways for OxLDL, the molecular details of OxLDL endocytosis by macrophages are not well understood. Studies in vitro demonstrate that the class B scavenger receptor CD36 mediates macrophage uptake and degradation of OxLDL. Although the closely related scavenger receptor class B type I (SR-BI) binds OxLDL with high affinity, evidence that SR-BI plays a role in OxLDL metabolism is lacking. In this study, we directly compared OxLDL uptake and degradation by CD36 and SR-BI. Our results indicate that although CD36 and SR-BI internalize OxLDL, SR-BI mediates significantly less OxLDL degradation. Endocytosis of OxLDL by both SR-BI and CD36 is independent of caveolae, microtubules, and actin cytoskeleton. However, OxLDL uptake by CD36, but not SR-BI, is dependent on dynamin. The analysis of chimeric SR-BI/CD36 receptors shows that the CD36 C-terminal cytoplasmic tail is necessary and sufficient for dynamin-dependent OxLDL internalization by class B scavenger receptors. These findings indicate that different mechanisms are involved in OxLDL uptake by SR-BI and CD36, which may segregate these two structurally homologous receptors at the cell surface, leading to differences in intracellular trafficking and degradation.     

Class B scavenger receptors CD36 and SR-BI are receptors for hypochlorite-modified low density lipoprotein

The presence of HOCl-modified epitopes inside and outside monocytes/macrophages and the presence of HOCl-modified apolipoprotein B in atherosclerotic lesions has initiated the present study to identify scavenger receptors that bind and internalize HOCl-low density lipoprotein (LDL). The uptake of HOCl-LDL by THP-1 macrophages was not saturable and led to cholesterol/cholesteryl ester accumulation. HOCl-LDL is not aggregated in culture medium, as measured by dynamic light scattering experiments, but internalization of HOCl-LDL could be inhibited in part by cytochalasin D, a microfilament disrupting agent. This indicates that HOCl-LDL is partially internalized by a pathway resembling phagocytosis-like internalization (in part by fluid-phase endocytosis) as measured with [14C]sucrose uptake. In contrast to uptake studies, binding of HOCl-LDL to THP-1 cells at 4 degrees C was specific and saturable, indicating that binding proteins and/or receptors are involved. Competition studies on THP-1 macrophages showed that HOCl-LDL does not compete for the uptake of acetylated LDL (a ligand to scavenger receptor class A) but strongly inhibits the uptake of copper-oxidized LDL (a ligand to CD36 and SR-BI). The binding specificity of HOCl-LDL to class B scavenger receptors could be demonstrated by Chinese hamster ovary cells overexpressing CD36 and SR-BI and specific blocking antibodies. The lipid moiety isolated from the HOCl-LDL particle did not compete for cell association of labeled HOCl-LDL to CD36 or SR-BI, suggesting that the protein moiety of HOCl-LDL is responsible for receptor recognition. Experiments with Chinese hamster ovary cells overexpressing scavenger receptor class A, type I, confirmed that LDL modified at physiologically relevant HOCl concentrations is not recognized by this receptor.     

Endothelial scavenger receptors

In the past few decades, cDNAs for endothelial scavenger receptors that bind to negatively charged molecules, particularly acetylated low density lipoproteins (Ac-LDL), have been cloned by expression cloning using modified LDL as ligands. A prototypic members of endothelial scavenger receptor family, namely, scavenger receptor class B type I (SR-BI) has been characterized as a high density lipoprotein (HDL) receptor. Another prototypic member, CD36, has been determined as a multiple ligand receptor because it binds to oxidized LDLs (Ox-LDL), trombospondin, erythrocytes infected with Plasmodium falciparum, long-chain fatty acids, and Gram-negative and Gram-positive bacteria. Lectin-like oxidized LDL receptor-1 (LOX-1) has been discovered as the principal receptor that mediates the action of Ox-LDL in the vascular walls. Recently, the structure of oxidized phospholipids, originally found in Ox-LDL, and its molecular mechanism of action on endothelial cells were determined. Further, the use of genetically manipulated rodent models and the recent forward genetic screening technique revealed the physiological and pathological functions of these endothelial scavenger receptors in innate immunity and infection. In this review, the structure and function of these multiligand scavenger receptors of endothelial cells have been described mainly in relation with lipid metabolism.     

ApoA-II modulates the association of HDL with class B scavenger receptors SR-BI and CD36

The class B scavenger receptors SR-BI and CD36 exhibit a broad ligand binding specificity. SR-BI is well characterized as a HDL receptor that mediates selective cholesteryl ester uptake from HDL. CD36, a receptor for oxidized LDL, also binds HDL and mediates selective cholesteryl ester uptake, although much less efficiently than SR-BI. Apolipoprotein A-II (apoA-II), the second most abundant HDL protein, is considered to be proatherogenic, but the underlying mechanisms are unclear. We previously showed that apoA-II modulates SR-BI-dependent binding and selective uptake of cholesteryl ester from reconstituted HDL. To investigate the effect of apoA-II in naturally occurring HDL on these processes, we compared HDL without apoA-II (from apoA-II null mice) with HDLs containing differing amounts of apoA-II (from C57BL/6 mice and transgenic mice expressing a mouse apoA-II transgene). The level of apoA-II in HDL was inversely correlated with HDL binding and selective cholesteryl ester uptake by both scavenger receptors, particularly CD36. Interestingly, for HDL lacking apoA-II, the efficiency with which CD36 mediated selective uptake reached a level similar to that of SR-BI. These results demonstrate that apoA-II exerts a marked effect on HDL binding and selective lipid uptake by the class B scavenger receptors and establishes a potentially important relationship between apoA-II and CD36.     

Scavenger receptors for oxidized and glycated proteins

Summary. Our present knowledge on chemically modified proteins and their receptor systems is originated from a proposal by Goldstein and Brown in 1979 for the receptor for acetylated LDL which is involved in foam cell formation, one of critical steps in atherogenesis. Subsequent extensive studies using oxidized LDL (OxLDL) as a representative ligand disclosed at least 11 different scavenger receptors which are collectively categorized as scavenger receptor family. Advanced glycation endproducts (AGE) and their receptor systems have been studied independently until recent findings that AGE-proteins are also recognized as active ligands by scavenger receptors including class A scavenger receptor (SR-A), class B scavenger receptors such as CD36 and SR-BI, type D scavenger receptor (LOX-1) and FEEL-1/FEEL-2. Three messages can be summarized from these experiments; (i) endocytic uptake of OxLDL and AGE-proteins by macrophages or macrophage-derived cells is mainly mediated by SR-A and CD36, which is an important step for foam cell formation in the early stage of atherosclerosis, (ii) selective uptake of cholesteryl esters of high density lipoprotein (HDL) mediated by SR-BI is inhibited by AGE-proteins, suggesting a potential pathological role of AGE in a HDL-mediated reverse cholesterol transport system, (iii) a novel scavenger receptor is involved in hepatic clearance of plasma OxLDL and AGE-proteins.     

Specific oxidized phospholipids inhibit scavenger receptor bi-mediated selective uptake of cholesteryl esters

We have recently demonstrated that specific oxidized phospholipids (oxPC(CD36)) accumulate at sites of oxidative stress in vivo such as within atherosclerotic lesions, hyperlipidemic plasma, and plasma with low high-density lipoprotein levels. oxPC(CD36) serve as high affinity ligands for the scavenger receptor CD36, mediate uptake of oxidized low density lipoprotein by macrophages, and promote a pro-thrombotic state via platelet scavenger receptor CD36. We now report that oxPC(CD36) represent ligands for another member of the scavenger receptor class B, type I (SR-BI). oxPC(CD36) prevent binding to SR-BI of its physiological ligand, high density lipoprotein, because of the close proximity of the binding sites for these two ligands on SR-BI. Furthermore, oxPC(CD36) interfere with SR-BI-mediated selective uptake of cholesteryl esters in hepatocytes. Thus, oxidative stress and accumulation of specific oxidized phospholipids in plasma may have an inhibitory effect on reverse cholesterol transport.     

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.     

Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptake in vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75-90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.     

Analysis of chimeric receptors shows that multiple distinct functional activities of scavenger receptor, class B, type I (SR-BI), are localized to the extracellular receptor domain

Scavenger receptor BI (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without degradation of the HDL particle. In addition, SR-BI stimulates the bi-directional flux of free cholesterol (FC) between cells and lipoproteins, an activity that may be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of FC from plasma HDL. SR-BI also increases cellular cholesterol mass and alters cholesterol distribution in plasma membrane domains as judged by the enhanced sensitivity of membrane cholesterol to extracellular cholesterol oxidase. In contrast, CD36, a closely related class B scavenger receptor, has none of these activities despite binding HDL with high affinity. In the present study, analyses of chimeric SR-BI/CD36 receptors and domain-deleted SR-BI have been used to test the various domains of SR-BI for functional activities related to HDL CE selective uptake, bi-directional FC flux, and the alteration of membrane cholesterol mass and distribution. The results show that each of these activities localizes to the extracellular domain of SR-BI. The N-terminal cytoplasmic tail and transmembrane domains appear to play no role in these activities other than targeting the receptor to the plasma membrane. The C-terminal tail of SR-BI is dispensable for activity as well for targeting to the plasma membrane. Thus, multiple distinct functional activities are localized to the SR-BI extracellular domain.     

CD36 does not play a direct role in HDL or LDL metabolism

CD36 and scavenger receptor class B, type I (SR-BI) are both class B scavenger receptors that recognize a broad variety of ligands, including oxidized low density lipoprotein (oxLDL), HDL, anionic phospholipids, and apoptotic cells. In this study we investigated the role of mouse CD36 (mCD36) as a physiological lipoprotein receptor. We compared the association of various lipoprotein particles with mCD36 and mSR-BI expressed in COS cells by adenovirus-mediated gene transfer. mCD36 bound human oxLDL and mouse HDL with high affinity. Human LDL bound poorly to mCD36, indicating that mCD36 is unlikely to play a significant role in LDL metabolism. The ability of mCD36 to mediate the selective uptake of cholesteryl esters (CE) from receptor-bound HDL was assessed. In comparison with mSR-BI, mCD36 inefficiently mediated the selective uptake of CE. Hepatic overexpression of mCD36 in C57BL/6 mice by adenovirus-mediated gene transfer did not result in significant alterations in plasma LDL and HDL levels. We conclude that mCD36, while able to bind HDL with high affinity, does not contribute significantly to HDL or LDL metabolism.     

SR-BI-directed HDL-cholesteryl ester hydrolysis

We have examined the metabolic fate of HDL cholesteryl ester (CE) delivered to cells expressing scavenger receptor class B type I (SR-BI). Comparison of SR-BI with a related class B scavenger receptor, CD36, showed a greater uptake and a more rapid and extensive hydrolysis of HDL-CE when delivered by SR-BI. In addition, hydrolysis of HDL-CE delivered by both receptors was via a neutral CE hydrolase. These data indicate that SR-BI, but not CD36, can efficiently direct HDL-CE to a neutral CE hydrolytic pathway. In contrast, LDL-CE was delivered and hydrolyzed equally well by SR-BI and CD36. Hydrolysis of LDL-CE delivered by SR-BI was via a neutral CE hydrolase but that delivered by CD36 occurred via an acidic CE hydrolase, indicating that SR-BI and CD36 deliver LDL-CE to different metabolic pathways. Comparison of inhibitor sensitivities in Y1-BS1 adrenal, Fu5AH hepatoma, and transfected cells suggests that hydrolysis of HDL-CE delivered by SR-BI occurs via cell type-specific neutral CE hydrolases. Furthermore, HDL-CE hydrolytic activity was recovered in a membrane fraction of Y1-BS1 cells. These findings suggest that SR-BI efficiently delivers HDL-CE to a metabolically active membrane compartment where CE is hydrolyzed by a neutral CE hydrolase.     

Uptake and fate of class B scavenger receptor ligands in HepG2 cells.

Class B scavenger receptors (SR-Bs) interact with native, acetylated and oxidized low-density lipoprotein (LDL, AcLDL and OxLDL), high-density lipoprotein (HDL3) and maleylated BSA (M-BSA). The aim of this study was to analyze the catabolism of CD36- and LIMPII-analogous-1 (CLA-1), the human orthologue for the scavenger receptor class B type I (SR-BI), and CD36 ligands in HepG2 (human hepatoma) cells. Saturation binding experiments revealed moderate-affinity binding sites for all the SR-B ligands tested with dissociation constants ranging from 20 to 30 microg.mL-1. Competition binding studies at 4 degrees C showed that HDL and modified and native LDL share common binding site(s), as OxLDL competed for the binding of 125I-LDL and 125I-HDL3 and vice versa, and that only M-BSA and LDL may have distinct binding sites. Degradation/association ratios for SR-B ligands show that LDL is very efficiently degraded, while M-BSA and HDL3 are poorly degraded. The modified LDL degradation/association ratio is equivalent to 60% of the LDL degradation ratio, but is three times higher than that of HDL3. All lipoproteins were good cholesteryl ester (CE) donors to HepG2 cells, as a 3.6-4.7-fold CE-selective uptake ([3H]CE association/125I-protein association) was measured. M-BSA efficiently competed for the CE-selective uptake of LDL-, OxLDL-, AcLDL- and HDL3-CE. All other lipoproteins tested were also good competitors with some minor variations. Hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes, whereas HDL3-CE was hydrolyzed in both lysosomal and extralysosomal compartments. Inhibition of the selective uptake of CE from HDL and native modified LDL by SR-B ligands clearly suggests that CLA-1 and/or CD36 are involved at least partially in this process in HepG2 cells.     

The role of human and mouse hepatic scavenger receptor class B type I (SR-BI) in the selective uptake of low-density lipoprotein-cholesteryl esters

Low-density lipoprotein (LDL)-cholesteryl ester (CE) selective uptake has been demonstrated in nonhepatic cells overexpressing the scavenger receptor class B type I (SR-BI). The role of hepatic SR-BI toward LDL, the main carrier of plasma CE in humans, remains unclear. The aim of this study was to determine if SR-BI, expressed at its normal level, is implicated in LDL-CE selective uptake in human HepG2 hepatoma cells and mouse hepatic cells, to quantify its contribution and to determine if LDL-CE selective uptake is likely to occur in the presence of human HDL. First, antibody blocking experiments were conducted on normal HepG2 cells. SR-BI/BII antiserum inhibited (125)I-LDL and (125)I-HDL(3) binding (10 microg of protein/mL) by 45% (p < 0.05) and CE selective uptake by more than 85% (p < 0.01) for both ligands. Second, HepG2 cells were stably transfected with a eukaryotic vector expressing a 400-bp human SR-BI antisense cDNA fragment. Clone 17 (C17) has a 70% (p < 0.01) reduction in SR-BI expression. In this clone, (3)H-CE-LDL and (3)H-CE-HDL(3) association (10 microg of protein/mL) was 54 +/- 6% and 45 +/- 7% of control values, respectively, while (125)I-LDL and (125)I-HDL(3) protein association was 71 +/- 3% and 58 +/- 5% of controls, resulting in 46% and 55% (p < 0.01) decreases in LDL- and HDL(3)-CE selective uptake. Normalizing CE selective uptake for SR-BI expression reveals that SR-BI is responsible for 68% and 74% of LDL- and HDL(3)-CE selective uptake, respectively. Thus, both approaches show that, in HepG2 cells, SR-BI is responsible for 68-85% of CE selective uptake. Other pathways for selective uptake in HepG2 cells do not require CD36, as shown by anti-CD36 antibody blocking experiments, or class A scavenger receptors, as shown by the lack of competition by poly(inosinic acid). However, CD36 is a functional oxidized LDL receptor on HepG2 cells, as shown by antibody blocking experiments. Similar results for CE selective uptake were obtained with primary cultures of hepatic cells from normal (+/+), heterozygous (-/+), and homozygous (-/-) SR-BI knockout mice. Flow cytometry experiments show that SR-BI accounts for 75% of DiI-LDL uptake, the LDL receptor for 14%, and other pathways for 11%. CE selective uptake from LDL and HDL(3) is likely to occur in the liver, since unlabeled HDL (total and apoE-free HDL(3)) and LDL, when added in physiological proportions, only partially competed for LDL- and HDL(3)-CE selective uptake. In this setting, human hepatic SR-BI may be a crucial molecule in the turnover of both LDL- and HDL(3)-cholesterol.     

High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae

Oxidized LDL (oxLDL) depletes caveolae of cholesterol, resulting in the displacement of endothelial nitric-oxide synthase (eNOS) from caveolae and impaired eNOS activation. In the present study, we determined if the class B scavenger receptors, CD36 and SR-BI, are involved in regulating nitric-oxide synthase localization and function. We demonstrate that CD36 and SR-BI are expressed in endothelial cells, co-fractionate with caveolae, and co-immunoprecipitate with caveolin-1. Co-incubation of cells with 10 microgram/ml high density lipoprotein (HDL) prevented oxLDL-induced translocation of eNOS from caveolae and restored acetylcholine-induced nitric-oxide synthase stimulation. Acetylcholine caused eNOS activation in cells incubated with 10 microgram/ml oxLDL (10-15 thiobarbituric acid-reactive substances) and blocking antibodies to CD36, whereas cells treated with only oxLDL were unresponsive. Furthermore, CD36-blocking antibodies prevented oxLDL-induced redistribution of eNOS. SR-BI-blocking antibodies were used to demonstrate that the effects of HDL are mediate by SR-BI. HDL binding to SR-BI maintained the concentration of caveola-associated cholesterol by promoting the uptake of cholesterol esters, thereby preventing oxLDL-induced depletion of caveola cholesterol. We conclude that CD36 mediates the effects of oxLDL on caveola composition and eNOS activation. Furthermore, HDL prevents oxLDL from decreasing the capacity for eNOS activation by preserving the cholesterol concentration in caveolae and, thereby maintaining the subcellular location of eNOS.     

Scavenger receptor class B type I-mediated reverse cholesterol transport is inhibited by advanced glycation end products

Cellular interactions of advanced glycation end products (AGE) are mediated by AGE receptors. We demonstrated previously that class A scavenger receptor types I and II (SR-A) and CD36, a member of class B scavenger receptor family, serve as the AGE receptors. In this study, we investigated whether scavenger receptor class B type I (SR-BI), another receptor belonging to class B scavenger receptor family, was also an AGE receptor. We used Chinese hamster ovary (CHO) cells overexpressed hamster SR-BI (CHO-SR-BI cells). (125)I-AGE-bovine serum albumin (AGE-BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-SR-BI cells. (125)I-AGE-BSA exhibited saturable binding to CHO-SR-BI cells (K(d) = 8.3 microg/ml). Endocytic uptake of (125)I-AGE-BSA by CHO-SR-BI cells was completely inhibited by oxidized low density lipoprotein (LDL) and acetylated LDL, whereas LDL exerted only a weak inhibitory effect (<20%). Cross-competition experiments showed that AGE-BSA had no effect on HDL binding to these cells and vice versa. Interestingly, however, SR-BI-mediated selective uptake of HDL-CE was completely inhibited by AGE-BSA in a dose-dependent manner (IC(50) <10 microg/ml). Furthermore, AGE-BSA partially inhibited (by <30%) the selective uptake of HDL-CE in human hepatocarcinoma HepG2 cells (IC(50) <30 microg/ml). In addition, [(3)H]cholesterol efflux from CHO-SR-BI cells to HDL was significantly inhibited by AGE-BSA in a dose-dependent manner (IC(50) <30 microg/ml). Our results indicate that AGE proteins, as ligands for SR-BI, effectively inhibit both SR-BI-mediated selective uptake of HDL-CE and cholesterol efflux from peripheral cells to HDL, suggesting that AGE proteins might modulate SR-BI-mediated cholesterol metabolism in vivo.     

TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages

Transforming growth factor-beta1 (TGF-beta1), a key cytokine for control of cell growth, extracellular matrix formation, and inflammation control, is secreted by many cells present in the arteriosclerotic plaque. Lipid accumulation in the vessel wall is regarded as an early step in atherogenesis and depends on uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors and their transformation into foam cells. Prominent members of the scavenger receptor family are the class A type I and II receptors (ScR-A), the class B receptor CD36, and the recently detected lectin-like oxidized LDL receptor-1 (LOX-1), which, unlike the native LDL receptor (LDL-R), are not feedback controlled. CD36 is responsible for >50% of modified LDL uptake into human monocyte-derived macrophages. We therefore studied whether TGF-beta1 influences expression and function of ScR-A, CD36, and LOX-1 in monocytes using RT-PCR and flow cytometry. Total uptake of oxidized LDL by monocytoid cells, reflecting the combined function of all scavenger receptors, was significantly reduced by TGF-beta1. At initially low picomolar concentrations, TGF-beta1 decreased CD36 mRNA and protein surface expression and ScR-A mRNA levels in the human monocytic cell line THP-1 and in freshly isolated and cultivated human monocytes, whereas LOX-1 mRNA was increased. Expression of LDL-R and beta-actin was not affected by TGF-beta1. In conclusion, depression of scavenger receptor function in monocytes by TGF-beta1 in low concentrations reduces foam cell formation. Together with matrix control by TGF-beta1, this may be important for atherogenesis and plaque stabilization.     

Structural Basis for the Recognition of Oxidized Phospholipids in Oxidized Low Density Lipoproteins by Class B Scavenger Receptors CD36 and SR-BI

Specific oxidized phospholipids (oxPC_CD36) accumulate in vivo at sites of oxidative stress and serve as high affinity ligands for scavenger receptors class B (CD36 and SR-BI). Recognition of oxPC_CD36 by scavenger receptors plays a role in several pathophysiological processes. The structural basis for the recognition of oxPC_CD36 by CD36 and SR-BI is poorly understood. A characteristic feature of oxPC_CD36 is an sn-2 acyl group that incorporates a terminal γ-hydroxy (or oxo)-α,β-unsaturated carbonyl. In the present study, a series of model oxidized phospholipids were designed, synthesized, and tested for their ability to serve as ligands for CD36 and SR-BI. We demonstrated that intact the sn-1 hydrophobic chain, the sn-3 hydrophilic phosphocholine or phosphatidic acid group, and the polar sn-2 tail are absolutely essential for high affinity binding. We further found that a terminal negatively charged carboxylate at the sn-2 position suffices to generate high binding affinity to class B scavenger receptors. In addition, factors such as polarity, rigidity, optimal chain length of sn-2, and sn-3 positions and negative charge at the sn-3 position of phospholipids further modulate the binding affinity. We conclude that all three positions of oxidized phospholipids are essential for the effective recognition by scavenger receptors class B. Furthermore, the structure of residues in these positions controls the affinity of the binding. The present studies suggest that, in addition to oxPC_CD36, other oxidized phospholipids observed in vivo may represent novel ligands for scavenger receptors class B.     

Role of scavenger receptors SR-BI and CD36 in selective sterol uptake in the small intestine

The serum lipoprotein high-density lipoprotein (HDL), which is a ligand of scavenger receptors such as scavenger receptor class B type I (SR-BI) and cluster determinant 36 (CD36), can act as a donor particle for intestinal lipid uptake into the brush border membrane (BBM). Both cholesterol and phospholipids are taken up by the plasma membrane of BBM vesicles (BBMV) and Caco-2 cells in a facilitated (protein-mediated) process. The protein-mediated transfer of cholesterol from reconstituted HDL to BBMV depends on the lipid composition of the HDL. In the presence of sphingomyelin, the transfer of cholesterol is slowed by a factor of about 3 probably due to complex formation between cholesterol and the sphingolipid. It is shown that the mechanism of lipid transfer from reconstituted HDL to either BBMV or Caco-2 cells as the acceptor is consistent with selective lipid uptake: the lipid donor docks at the membrane-resident scavenger receptors which mediate the transfer of lipids between donor and acceptor. Selective lipid uptake implies that lipid, but no apoprotein is transferred from the donor to the BBM, thus excluding endocytotic processes. The two BBM models used here clearly indicate that fusion of donor particles with the BBM can be ruled out as a major mechanism contributing to intestinal lipid uptake. Here we demonstrate that CD36, another member of the family of scavenger receptors, is present in rabbit and human BBM vesicles. This receptor mediates the uptake of free cholesterol, but not of esterified cholesterol, the uptake of which is mediated exclusively by SR-BI. More than one scavenger receptor appears to be involved in the uptake of free cholesterol with SR-BI contributing about 25% and CD36 about 35%. There is another yet unidentified protein accounting for the remaining 30 to 40%.     

Class B scavenger receptor types I and II and CD36 targeting improves sepsis survival and acute outcomes in mice

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.