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Identification of the human analog of SR-BI and LOX-1 as receptors for hypochlorite-modified high density lipoprotein on human umbilical venous endothelial cells^{1 ,2}

GUNTHER MARSCHE^*, SANJA LEVAK-FRANK^*, OSWALD QUEHENBERGER, REGINE HELLER, WOLFGANG SATTLER^* and ERNST MALLE^*3

^* Institute of Medical Biochemistry, Karl-Franzens University, Graz, Austria;
Department of Medicine, University of California, San Diego, La Jolla, California, USA; and
Friedrich-Schiller University Jena, Center of Vascular Biology and Medicine, Erfurt, Germany

³Correspondence: Karl-Franzens University Graz, Institute of Medical Biochemistry, Harrachgasse 21, A-8010 Graz, Austria. E-mail: ernst.malle{at}kfunigraz.ac.at

SPECIFIC AIMS

Theoxidation theory of atherosclerosis proposes that oxidation of lipoproteins contributes to atherogenesis probably via endothelial activation and/or subsequent endothelial dysfunction. One of the potential in vivo oxidants able to modify/oxidize (lipo)proteins is hypochlorous acid/hypochlorite (HOCl/OCl^-) generated by the myeloperoxidase (MPO)-H₂O₂-halide system of activated phagocytes. Colocalization of MPO and HOCl-modified (lipo)proteins in human lesion material and endothelial cells prompted us to investigate pathways that are responsible for the interaction of HOCl-modified high density lipoprotein (HOCl-HDL₃) with human umbilical venous endothelial cells (HUVECs).

PRINCIPAL FINDINGS

1. HUVECs mediate holoparticle- and selective cholesterylester (CE) uptake from HOCl-HDL₃
Binding of ¹²⁵I-HDL₃ to HUVECs at 4°C was saturable (K_d: 27 µg HDL₃ protein/ml, b_max: 170 ng HDL₃ protein/mg cell protein) whereas binding of ¹²⁵I-HOCl-HDL₃ (HOCl: HDL₃ molar ratio of 200) was not saturable within the concentrations applied (K_d: 231 µg HOCl-HDL₃ protein/ml, b_max: 639 ng HOCl-HDL₃ protein/mg cell protein). Competition experiments performed at 4°C revealed that binding of ¹²⁵I-labeled HDL₃/HOCl-HDL₃ was effectively competed only by autologous lipoprotein particles. Next, HDL-holoparticle turnover was assessed as a function of the degree of HOCl modification (Fig. 1A ). During these experiments it became obvious that the bound and internalized lipoprotein fraction increased in a near linear fashion dependent on the degree of HOCl modification. A pronounced effect was observed when degradation rates of lipoprotein particles (which were also dependent on the degree of HOCl modification) were measured. To study the selective CE uptake, HDL₃ and HOCl-HDL₃ were labeled in the protein (¹²⁵INa) and the lipid moiety (using cholesteryl-1,2,6,7-[³H]-palmitate), and the cell association of both tracers was analyzed. Similar as shown in Fig. 1A holoparticle association of ¹²⁵I-labeled lipoproteins increased as a function of the degree of HOCl modification (Fig. 1B ). This was also reflected by total [³H]CE uptake. For both HDL₃ and HOCl-HDL₃, a high selective CE uptake became apparent, exceeding holoparticle uptake on average by sevenfold.

View larger version (13K): [in this window] [in a new window]   Figure 1. Binding properties of HDL3 and HOCl-HDL3 (HOCl: HDL3 molar ratio of 50, 100, and 200) by HUVECs at 37°C. A) 125I-labeled lipoprotein particles; specific binding (), internalization (), and degradation (•). B) Selective CE uptake () calculated as the difference of cell association between [3H]CE (•) and 125I-labeled lipoprotein particles ().

2. Identification of human scavenger receptor class B, type I (hSR-BI), a mediator for selective CE uptake of HOCl-HDL₃ on HUVECs
Both the high selective CE uptake (Fig. 1B ) and competition experiments using phosphatidylserine (PS) -containing vesicles (data not shown) suggested that hSR-BI is a candidate to mediate CE uptake from HOCl-HDL₃. By RT-PCR using hSR-BI-specific primers, a 553 bp product was amplified from total RNA from HUVECs. Northern blot analysis using the radiolabeled 553-bp fragment for hybridization confirmed the presence of mRNA for hSR-BI in HUVECs. Immunoblotting experiments of detergent solubilized membrane protein fractions from HUVECs revealed the 84 kDa hSR-BI protein. To assess whether SR-BI mediates selective CE uptake from HOCl-HDL₃, CHO cells overexpressing SR-BI (ldlA[mSR-BI]) were used. An increased cell association of [³H]CE-labeled HDL₃ and HOCl-HDL₃ by ldlA[mSR-BI] cells ( sevenfold higher than observed with ldlA7 cells used as controls) revealed both lipoprotein preparations as SR-BI ligands. CE association of both lipoprotein particles to ldlA[mSR-BI] was competed by PS-containing vesicles (known SR-BI ligands).

3. Lectin-like oxidized LDL receptor (LOX-1) on HUVECs mediates holoparticle uptake and degradation of HOCl-HDL₃
As degradation rates of HOCl-HDL₃ by HUVECs increased as a function of increasing degree of HOCl modification and could be inhibited up to 35% by chloroquine (100 µM), we studied whether scavenger receptor expressed on endothelial cells (SREC) and/or LOX-1 contribute to enhanced holoparticle turnover of HOCl-HDL₃. As SREC has been reported to mediate binding and degradation of acetylated low density lipoprotein (Ac-LDL), competition experiments with different ligands (50-fold molar excess) were performed. Only unlabeled Ac-LDL was observed to inhibit cell association of ¹²⁵I-Ac-LDL by HUVECs whereas LDL, Cu²⁺-oxidized LDL (Cu²⁺-ox-LDL) or HOCl-HDL₃ (at different HOCl: HDL₃ molar ratios) exhibited no remarkable displacement capability. However, the cell association of ¹²⁵I-Cu²⁺-ox-LDL (a preferential ligand for LOX-1) was inhibited by Cu²⁺-ox-LDL, Ac-LDL, and HOCl-HDL₃, respectively; native HDL₃ and LDL did not compete (Fig. 2A ).

View larger version (53K): [in this window] [in a new window]   Figure 2. Identification of LOX-1 on HUVECs mediating binding and cell association of HOCl-HDL3. A) Competition experiments of 5 µg/ml 125I-Cu2+-ox-LDL in the absence or presence of indicated competitors. B) Membrane protein fractions isolated from PMA-stimulated (1 to 4) and resting cells (5 and 6). Immunochemical detection of LOX-1 (2 and 5). Ligand blots were performed by incubating the membranes with 50 µg/ml HDL3 (1) and HOCl-HDL3 (HOCl: HDL3 molar ratio of 200; 3 and 6). The nitrocellulose was preincubated with anti-LOX-1 IgG (4) prior addition of 50 µg/ml HOCl-HDL3. Anti-apo A-I IgG (1, 3, 4, and 6) was used as primary antibody.

To further investigate whether LOX-1 is involved in binding of HOCl-HDL₃, detergent solubilized membrane protein fractions from resting and PMA-stimulated (known to induce LOX-1 protein expression) HUVECs were used. Indeed, LOX-1 could be identified on HUVECs (Fig. 2B ). Ligand blots revealed a major binding protein for HOCl-HDL₃ (but not for HDL₃) comigrating with the 35 kDa protein band identified with anti-LOX-1 IgG. Preincubation of the nitrocellulose with anti-LOX-1 IgG for 2 h and subsequent ligand blot experiments revealed impaired immunoreactivity for HOCl-HDL₃, indicating that HOCl-HDL₃ and anti-LOX-1 IgG bind to the same receptor. The same results were obtained when HUVECs were stimulated with angiotensin II. Under nonstimulated conditions, only a faint band could be identified with anti-LOX-1 IgG, but no binding of HOCl-HDL₃ to the 35 kDa protein could be observed (Fig. 2B ).

CONCLUSIONS

Besides affecting endothelial function via redox-dependent/sensitive pathways, HOCl (generated by the MPO-H₂O₂-halide system) or added as reagent causes conversion of HDL₃ into a phagocytosable particle for macrophages and alters cellular cholesterol-efflux capacity of HDL₃. The major findings obtained here with HUVECs were 1) the identification of hSR-BI on HUVECs, 2) the observation that HOCl-HDL₃ is a ligand for hSR-BI, 3) the recognition of HOCl-HDL₃ by hSR-BI is coupled to efficient selective uptake of CE, and 4) the observation that LOX-1 binds HOCl-HDL₃ and presumably mediates endothelial internalization and subsequent degradation.

Our results indicate that LOX-1 and hSR-BI are both involved in the metabolism of HOCl-HDL₃, seemingly by different pathways (Fig. 3 ). From the present data it appears that an increasing oxidant:HDL₃ molar ratio results in altered degradation rates, probably as a threshold event as described for clearance of asialoglycoproteins or the recognition pattern of monocyte-derived macrophages for malondialdehyde-modified LDL. Whereas a low oxidant:lipoprotein molar ratio resulted in decreased degradation rates, a high oxidant excess markedly accelerated degradation rates of HOCl-HDL₃. The fact that degradation rates were chloroquine-sensitive only for HOCl-HDL₃ suggested lysosomal degradation as a consequence of scavenger-receptor-mediated uptake mechanisms. Although we cannot provide evidence for the receptor responsible for internalization of HDL₃, ligand blots in combination with antibody inhibition and competition experiments made LOX-1 the most likely candidate for internalization and subsequent degradation of HOCl-HDL₃ holoparticles. LOX-1 expression is not restricted to cultured endothelial cells but was also observed in human and rabbit atherosclerotic lesions. In addition, LOX-1 expression is inducible by angiotensin II, certain cytokines, and modified phospholipids that are relevant to atherogenesis. Apoptotic cells and ox-LDL are probably the physiologically most relevant ligands for LOX-1 in atherosclerotic lesions. LOX-1-mediated internalization of ox-LDL and its oxidized lipid constituents that are present in atherosclerotic plaque could impair endothelial production of NO or induce the endothelial expression of leukocyte adhesion molecules and/or growth factors involved in atherogenesis. Thus, uptake of ox-LDL by LOX-1 in vascular endothelium may cause endothelial activation and/or dysfunction. In line with the relatively broad ligand specificity of SR-BI, selective CE uptake from HOCl-HDL₃ was observed during the present study. One question arising from this observation is whether this pathway displays beneficial or deleterious effects. This is most probably related to the severity of inflammatory conditions and the resulting oxidant:lipoprotein molar ratio. Our in vitro studies have shown that at HOCl: HDL₃ molar ratios of 60, apo A-I represents the preferential target for HOCl attack with the lipid domain remaining unaffected. Under these conditions, the recognition of modified HDL₃ by extrahepatic tissues could indicate that these tissues can still receive the required amount of native/modified cholesterol via selective uptake of CE by SR-BI. On the other hand, apolipoprotein-located chloramines can induce lipid peroxidation in HOCl-modified lipoproteins in a time-dependent manner by secondary derived radicals and could lead to selective uptake of ox-CEs. It is conceivable that this effect could also occur during MPO-mediated oxidation of lipoproteins deposited in atherosclerotic tissues. In vitro studies using HOCl either as reagent or generated by the MPO-H₂O₂ chloride system have demonstrated that the major lipid classes present in lipoproteins are attacked by HOCl and converted to the corresponding chlorinated and/or oxidized derivates. In particular, it was suggested that cholesterol chlorohydrins could exert powerful biological effects in the artery wall. Whether or not CE-depleted HOCl-HDL₃, a lipoprotein particle containing chloramines (interesting oxidants with signal-modulating functions), is subsequently cleared by LOX-1 and whether this and/or other pathways are involved in endothelial dysfunction (Fig. 3) remain to be elucidated.

View larger version (25K): [in this window] [in a new window]   Figure 3. Interaction of HOCl-HDL with endothelial cells. Apical events: circulating native HDL might be a preferential ligand for SR-BI (1) resulting in selective delivery of HDL constituents to endothelial cells. Next, native HDL can be modified by the MPO-H2O2-halide system of activated leukocytes that adhere to the endothelial layer (2). Subsequent interaction with SR-BI (1) might be a selective delivery pathway for modified lipids. Holoparticle uptake of HOCl-HDL (3) or ox-CE/CE-depleted HOCl-HDL (3‘) may occur via LOX-1. Basolateral events: after migration across the endothelial layer (4), MPO-dependent pathways contribute to HDL modification (2‘). HOCl-HDL may either escape from the intima (5) or generate lipid-enriched macrophages via lipid uptake (1) or holoparticle uptake (3"). The different degree of HOCl modification will affect cholesterol efflux properties (6) of the modified lipoprotein particle and reverse cholesterol transport (5‘). The combined effects of HOCl-modified particles (modified protein and lipid moieties) could ultimately result in an imbalance of basolateral signaling pathways and/or endothelial dysfunction (7).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0532fje ; to cite this article, use FASEB J. (February 26 2001) 10.1096/fj.00-0532fje

² The authors are grateful to Dr. M. Krieger (MIT) providing ldlA[mSR-BI] and ldlA7 cells. The expert technical assistance of B. Hirschmugl is appreciated. This work was supported by the DFG (HE 2304/1–1), OENB (8778 and 8127), and the FWF (P14109 and P14186).

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