Objective-The ability of the potent cholesteryl ester transfer protein (CETP) inhibitor torcetrapib 414) to raise high-density lipoprotein cholesterol (HDL-C) levels in healthy young subjects was tested in this initial phase 1 multidose study. Methods and Results-Five groups of 8 subjects each were randomized to placebo (nϭ2) or torcetrapib (nϭ6) at 10, 30, 60, and 120 mg daily and 120 mg twice daily for 14 days. Torcetrapib was well tolerated, with all treated subjects completing the study. The correlation of plasma drug levels with inhibition (EC50ϭ43 nM) was as expected based on in vitro potency (IC50 Ϸ50 nM), and increases in CETP mass were consistent with the proposed mechanism of inhibition. CETP inhibition increased with escalating dose, leading to elevations of HDL-C of 16% to 91%. Total plasma cholesterol did not change significantly because of a reduction in nonHDL-C, including a 21% to 42% lowering of low-density lipoprotein cholesterol at the higher doses. Apolipoprotein A-I and E were elevated 27% and 66%, respectively, and apoB was reduced 26% with 120 mg twice daily. Cholesteryl ester content decreased and triglyceride increased in the nonHDL plasma fraction, with contrasting changes occurring in HDL. studies, the inverse correlation between high-density lipoprotein (HDL) levels and premature coronary heart disease (CHD) has been strengthened. A 1% decrease in HDL cholesterol (HDL-C) has been associated with a 1% to 2% increase in risk for CHD, 3 and lipid intervention trials have demonstrated that increases in HDL-C 4 and its main apoprotein, A-I, 5 contribute to reduced CHD, even in the absence of any change in low-density lipoprotein cholesterol (LDL-C). 6 However, current therapies for raising HDL are limited. The fibrates and statins produce only modest elevations in HDL, and the use of niacin, although somewhat more effective, has been hampered by side effects. 7 In a recent study, the combination of lovastatin and extended-release niacin (Advicor) was able to increase HDL levels by 20% to 32%, 8 but withdrawal rates for incidents of flushing and other adverse events were relatively high. Conclusions-TheseThe marked increase in HDL associated with human deficiency of cholesteryl ester transfer protein (CETP) 9 has See page 387 and cover suggested CETP inhibition as a means of elevating HDL. Expression of CETP in transgenic mice under different metabolic settings has produced mixed results regarding its atherogenicity, whereas inhibition of endogenous CETP in rabbits has more consistently been antiatherogenic. 10 With regard to human CETP mutations and the associated reduction in CETP levels, recent analysis of prospective data from the Honolulu Heart Study 10 is consistent with the results of a previous study of Japanese subjects 11 in concluding that CETP deficiency is protective when associated with HDL-C levels Ն60 mg/dL.The results for a phase 2 study testing the synthetic CETP inhibitor JTT-705 in subjects with mild hyperlipidemia have been reported. 12 At 900 mg daily, JTT-705 led to ...

Cholesteryl ester transfer protein (CETP) inhibitors increase high density lipoprotein-cholesterol (HDL-C) in animals and humans, but whether CETP inhibition will be antiatherogenic is still uncertain. We tested the CETP inhibitor torcetrapib in rabbits fed an atherogenic diet at a dose sufficient to increase HDL-C by at least 3-fold (207 6 32 vs. 57 6 6 mg/dl in controls at 16 weeks). CETP activity was inhibited by 70-80% throughout the study. Non-HDL-C increased in both groups, but there was no difference apparent by the study's end. At 16 weeks, aortic atherosclerosis was 60% lower in torcetrapib-treated animals (16.4 6 3.4% vs. 39.8 6 5.4% in controls) and aortic cholesterol content was reduced proportionally. Sera from a separate group of rabbits administered torcetrapib effluxed 48% more cholesterol from Fu5AH cells than did sera from control animals, possibly explaining the reduced aortic cholesterol content. Regression analyses indicated that lesion area in the torcetrapib-treated group was strongly correlated with the ratio of total plasma cholesterol to HDL-C but not with changes in other lipid or lipoprotein levels. CETP inhibition with torcetrapib retards atherosclerosis in rabbits, and the reduced lesion area is associated with increased levels of HDL-C. High levels of high density lipoprotein-cholesterol (HDL-C) have been associated with a decreased incidence of coronary heart disease in epidemiological studies (1-3), and HDL has a number of potentially antiatherogenic properties that may account for its atheroprotective effects.HDL is a key mediator of reverse cholesterol transport, the process by which excess peripheral tissue cholesterol is shunted back to the liver. However, HDL has antiinflammatory (4, 5), antioxidative (6, 7), and antithrombotic activities (8, 9) that may also contribute to its antiatherogenic effects. Because our understanding of exactly how HDL protects against atherosclerosis is not complete, and because HDL speciation, metabolism, and function are complex, perhaps not all mechanisms for increasing HDL-C will ultimately be shown to have equivalent effects on the atherosclerotic process. One HDL-increasing target that has already triggered debate in this regard is cholesteryl ester transfer protein (CETP).CETP is a plasma glycoprotein that transfers cholesteryl esters (CEs), triglycerides, and phospholipids among circulating lipoproteins (10, 11). CETP transfers neutral lipids down concentration gradients; as such, the physiologically relevant direction of the transfer of CE is from the CE-enriched HDL fraction to non-HDL lipoproteins, with retrograde transfer of triglycerides. In the context of reverse cholesterol transport, the transfer of CE via CETP can divert HDL-CE from the direct, hepatic, specific uptake pathway to an indirect pathway for hepatic CE delivery involving the receptor-mediated uptake of apolipoprotein B (apoB)-containing lipoproteins. Under conditions in which hepatic apoB uptake is downregulated, CETP action results in a CE enrichment of non-H...

tions in PCSK9 cause hypercholesterolemia in humans ( 1-4 ), whereas loss-of-function mutations in humans lead to a reduction in LDL cholesterol and a marked decrease in the risk of coronary heart disease ( 5-7 ). PCSK9 is a member of the subtilisin serine protease family and the proteinase K subfamily. The protein is synthesized as a proprotein that autocatalytically cleaves to generate mature protein ( 8 ). PCSK9 protein is secreted as a complex containing cleaved N-terminal prodomain, which remains associated with the catalytic domain ( 8 ). The catalytic activity is required for PCSK9 maturation and secretion ( 9 ) but appears not to be essential for the reduction of LDLR (LDL receptor) by secreted PCSK9 ( 10 ).PCSK9 is predominantly expressed in liver, intestine, and to a lesser extent, in kidney ( 8 ). PCSK9 gene expression is regulated by cholesterol via sterol regulatory element-binding protein (SREBP) pathways ( 11,12 ). In mice, PCSK9 gene expression is downregulated by dietary cholesterol and dramatically upregulated by hepatic overexpression of the transactivation domain of SREBP-1a and SREBP-2 ( 12 ). Deletion of PCSK9 in mice leads to elevated liver LDLR protein (but not mRNA) and to reduced plasma total cholesterol, mainly HDL cholesterol ( 13 ). Furthermore, PCSK9 defi ciency increases LDL clearance and enhances the response to statin ( 13 ). Knockdown of PCSK9 by antisense approach also decreases total cholesterol in high-fat-fed mice in an LDLR dependent manner ( 14 ). Hepatic overexpression of murine or human PCSK9 by adenovirus ( 9, 15, 16 ) or transgene ( 17 ) results in the reduction of liver LDLR protein and elevation of LDL cholesterol. Transgenic mice overexpressing PCSK9 in liver accumulated PCSK9 to a level of 100-400 g/ml in plasma and reduced liver LDLR protein in wild-type mice that are parabiotically Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) is predominantly expressed in liver and regulates cholesterol metabolism by down regulating liver LDL receptor (LDLR) proteins. Here we report transgenic overexpression of human PCSK9 in kidney increased plasma levels of PCSK9 and subsequently led to a dramatic reduction in liver LDLR proteins. The regulation of LDLR by PCSK9 displayed tissue specifi city, with liver being the most responsive tissue. Even though the PCSK9 transgene was highly expressed in kidney, LDLR proteins were suppressed to a lower extent in this tissue than in liver. Adrenal LDLR proteins were not regulated by elevated plasma PCSK9. hPCSK9 transgene expression and subsequent reduction of liver LDLR led to increases in plasma total cholesterol, LDL cholesterol, and ApoB, which were further increased by a highfat, high-cholesterol diet. We also observed that the size distribution of hPCSK9 in transgenic mouse plasma was heterogeneous. In chow-fed mice, the majority of PCSK9 proteins were in free forms; however, feeding a high-fat, high-cholesterol diet resulted in a shift of hPCSK9 distribution toward larger complexes. PCSK9 distribution in human plasma...

Lectin-like oxidized LDL (ox-LDL) receptor-1 (LOX-1) is a type-II transmembrane protein that belongs to the C-type lectin family of molecules. LOX-1 acts as a cell surface endocytosis receptor and mediates the recognition and internalization of ox-LDL by vascular endothelial cells. Internalization of ox-LDL by LOX-1 results in a number of pro-atherogenic cellular responses implicated in the development and progression of atherosclerosis. In an effort to elucidate the functional domains responsible for the binding of ox-LDL to the receptor, a series of site-directed mutants were designed using computer modeling and X-ray crystallography to study the functional role of the hydrophobic tunnel present in the LOX-1 receptor. The isoleucine residue (I 149 ) sitting at the gate of the channel was replaced by phenylalanine, tyrosine, or glutamic acid to occlude the channel opening and restrict the docking of ligands to test its functional role in the binding of ox-LDL. The synthesis, intracellular processing, and cellular distribution of all mutants were identical to those of wild type, whereas there was a marked decrease in the ability of the mutants to bind ox-LDL. These studies suggest that the central hydrophobic tunnel that extends through the entire LOX-1 molecule is a key functional domain of the receptor and is critical for the recognition of modified LDL. Lectin-like oxidized LDL receptor-1 (LOX-1) is a member of the class E scavenger receptor family, a structurally diverse group of cell surface receptors of the innate immune system that recognize and internalize oxidized LDL (ox-LDL) in endothelial cells of large arteries (1). More recent studies have indicated that LOX-1 is expressed in other cells types, including macrophages (2), vascular smooth-muscle cells (3), and platelets (4). Its expression is not constitutive, but rather, markedly induced by proinflammatory, oxidative, and mechanical stimuli (5, 6), which leads to the activation of endothelial cells, transformation of smooth-muscle cells, and accumulation of lipids in macrophages, resulting in cellular injury and the development of atherosclerosis. Studies in animal models have provided further evidence in support of a role for LOX-1 in atherosclerosis. Overexpression of LOX-1 in mice leads to the formation of atheroma-like lesion areas (7). Conversely, its deletion sustains endothelial function and confers protection in the development of atherosclerosis in association with decreased inflammatory and pro-oxidant markers (8). Finally, human genetic studies strengthen the role of this receptor in cardiovascular disease (9-11).LOX-1 is a disulfide-linked homodimeric type II transmembrane protein with a short 34-residue cytoplasmic tail, a single transmembrane domain, and an extracellular region consisting of an 80-residue domain predicted to be a coil followed by a 130-residue C-terminal C-type lectinlike domain (CTLD) responsible for ox-LDL recognition (12)(13)(14). Homodimers are formed via an interchain disulfide bond between Cys 140 residu...

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids from triglyceride-rich lipoproteins into HDL. PLTP has been shown to be an important factor in lipoprotein metabolism and atherogenesis. Here, we report that chronic high-fat, high-cholesterol diet feeding markedly increased plasma cholesterol levels in C57BL/6 mice. PLTP deficiency attenuated diet-induced hypercholesterolemia by dramatically reducing apolipoprotein E-rich lipoproteins (288%) and, to a lesser extent, LDL (240%) and HDL (235%). Increased biliary cholesterol secretion, indicated by increased hepatic ABCG5/ABCG8 gene expression, and decreased intestinal cholesterol absorption may contribute to the lower plasma cholesterol in PLTP-deficient mice. The expression of proinflammatory genes (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) is reduced in aorta of PLTP knockout mice compared with wild-type mice fed either a chow or a high-cholesterol diet. Furthermore, plasma interleukin-6 levels are significantly lower in PLTP-deficient mice, indicating reduced systemic inflammation. These data suggest that PLTP appears to play a proatherogenic role in dietinduced hyperlipidemic mice. Plasma phospholipid transfer protein (PLTP) plays an important role in the metabolism of lipoproteins (1). PLTP belongs to the family of lipid transfer/lipopolysaccharide binding proteins, including cholesteryl ester transfer protein, lipopolysaccharide binding protein, and bactericidal permeability-increasing protein (2, 3). It has been shown that PLTP facilitates the transfer and exchange of phospholipids between VLDL and HDL (4). It also transfers phospholipids between HDL particles that result in the conversion of HDL 3 into larger and smaller HDL particles (5, 6). PLTP can also bind several other amphipathic molecules, including a-tocopherol, diacylglycerides, cerebrosides, and lipopolysaccharides (7). Several clinical studies suggest that high plasma PLTP activity is a risk factor for coronary artery disease and a determinant of carotid intimamedia thickness in type 2 diabetes mellitus (8, 9).Studies using genetically modified mice strongly suggest that PLTP functions as a proatherogenic factor (10-12). PLTP deficiency causes a marked decrease in HDL lipids and apolipoprotein A-I (apoA-I), as a result of higher catabolism, in both chow-fed and 2 week Western diet-fed mice (13,14). The absence of PLTP in hyperlipidemic apoE-deficient and human apoB transgenic mouse strains results in reduced production in plasma levels of apoBcontaining lipoproteins, mostly LDL (10). Atherosclerotic lesion areas are also decreased in PLTP knockout mice in the LDL receptor-or apoE-deficient or apoB transgenic background, despite decreased HDL. Furthermore, reduced lipoprotein oxidation and improved anti-inflammatory properties of HDL in PLTP knockout mice may also contribute to the antiatherogenic potential in these mouse models (15,16). The proatherogenic role of PLTP is further supported by results that demonstrate increased ...