The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-β-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-β-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [3H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [3H]neutral sterols and [3H]bile acids, whereas all compounds increased plasma HDL-[3H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-β-HDL formation, which may be required to increase reverse cholesterol transport.

Abstract-A novel lectinlike oxidized low density lipoprotein receptor-1 (LOX-1) was recently identified in bovine aortic endothelial cells. It is strongly suggested to have a potential role in the initiation and development of atherosclerosis.In this study, we have isolated cDNA clones encoding the rabbit homologue of LOX-1 by screening a rabbit placenta cDNA library. In amino acid sequence and domain structure organization, the rabbit LOX-1 is highly conserved with the human counterpart. Transfection of rabbit LOX-1 cDNA to HEK-293 cells confers on them the activity to bind and internalize oxidized low density lipoprotein. Rabbit LOX-1 was identified as a 45-kDa protein by Western blot analysis with a specific monoclonal antibody. Notably, analyses by reverse transcription-polymerase chain reaction and Western blot revealed that LOX-1 was accumulated in 8-week-old Watanabe heritable hyperlipidemic rabbit aortas compared with normal rabbit aortas. Immunostaining confirmed that the augmented expression of LOX-1 was primarily localized within the intima at the earliest stages of atherogenesis. The most prominent staining was in the endothelial cells of lesions. Furthermore, the distinctive staining of LOX-1 was identified in the endothelium of nonlesion areas of Watanabe heritable hyperlipidemic rabbit aortas. Taken together, these findings support the possibility that LOX-1 might be involved in the initiation of atherosclerosis. Key Words: atherosclerosis Ⅲ endothelium Ⅲ oxidized low density lipoproteins Ⅲ lectinlike oxidized low density lipoprotein receptor-1 Ⅲ Watanabe heritable hyperlipidemic rabbits E ndothelial dysfunction is implicated in the pathogenesis of atherosclerosis. 1-3 Oxidized LDL (Ox-LDL) is believed to be a key atherogenic component relevant to endothelial injury in vivo. 1,4 A number of "scavenger receptors" characterized by binding to Ox-LDL have been identified. 5,6 Among them, the lectinlike Ox-LDL receptor-1 (LOX-1) is expressed in the endothelial cells of large arteries. Applying an expression cloning strategy, we originally identified the LOX-1 cDNA from bovine aortic endothelial cells. 7 LOX-1 is a type II membrane protein with a C-type lectinlike structure at the C-terminus. In addition to Ox-LDL, LOX-1 binds aged/apoptotic cells, suggesting potential physiological functions. 8 LOX-1 gene expression is highly regulated. The inducible expression in cultured endothelial cells by phorbol ester, tumor necrosis factor-␣, angiotensin II, and shear stress, as well as the ligand, Ox-LDL, has been reported. 7,9 -13 Besides these in vitro studies, the expression of LOX-1 was confirmed in normal arteries and atherosclerotic intima in vivo. 7,14 -16 An enhanced expression in hypertensive rat aorta was also observed. 14,15 Integrating inflammatory and fluid mechanical stimuli, the initiation of atherosclerosis develops focally and is accelerated under hypertension. This inducible nature of LOX-1 expression suggests an active role in the complex atherogenic processes. 17 The rabbit is an important ...

One crucial role of endothelium is to keep the innermost surface of a blood vessel antithrombotic. However, the endothelium also expresses prothrombotic molecules in response to various stimuli. The balance between the antithrombotic and prothrombotic nature of the endothelium is lost under certain conditions. During atherosclerosis, the attachment of platelets to the vessel surface has been suggested to promote the proliferation of smooth muscle cells and intimal thickening as well as to affect the prognosis of the disease directly through myocardial infarction and stroke. Dysfunctional endothelium, which is often a result of the action of oxidized low-density lipoprotein (OxLDL), tends to be more procoagulant and adhesive to platelets. Herein, we sought the possibility that the endothelial lectin-like OxLDL receptor-1 (LOX-1) is involved in the platelet-endothelium interaction and hence directly in endothelial dysfunction. LOX-1 indeed worked as an adhesion molecule for platelets. The binding of platelets was inhibited by a phosphatidylserine-binding protein, annexin V, and enhanced by agonists for platelets. These results suggest that negative phospholipids exposed on activation on the surface of platelets are the epitopes for LOX-1. Notably, the binding of platelets to LOX-1 enhanced the release of endothelin-1 from endothelial cells, supporting the induction of endothelial dysfunction, which would, in turn, promote the atherogenic process. LOX-1 may initiate and promote atherosclerosis, binding not only OxLDL but also platelets. O xidized low-density lipoprotein (OxLDL) is believed to be an essential atherogenic component that induces endothelial dysfunction and accumulation of foam cells (1). A number of ''scavenger receptors'' characterized by binding to OxLDL have been identified (2). Among them, lectin-like OxLDL receptor-1 (LOX-1), identified in our laboratory, is uniquely expressed in the endothelial cells of large arteries (3). LOX-1 is a type II membrane protein with a C-type lectin-like structure at the C terminus. The expression of LOX-1 in endothelial cells in vitro and in vivo is highly regulated. LOX-1 expression is induced by tumor necrosis factor-␣, phorbol ester, shear stress, lipopolysaccharide, angiotensin II, and OxLDL in cultured endothelial cells as well as by hypertension in vivo (4-10). Recently, LOX-1 was found to be expressed in macrophages in atheromatous intima and culture as well as in vascular smooth muscle cells in atheromatous intima (5,11,12). Because macrophages and smooth muscle cells transform into foam cells in atheroma, a potential role for LOX-1 in foam cell formation has been suggested.Besides OxLDL, LOX-1 binds aged͞apoptotic cells, suggesting potential physiological function (13). Some of the other receptors for OxLDL have also been reported to bind apoptotic cells, although the recognition mechanisms are not fully clarified (2). In the study of the mechanisms responsible for the binding of apoptotic cells, we have found that anionic phospholipids are involved in the...

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