Cholesteryl ester transfer protein genotypes associated with venous thrombosis and dyslipoproteinemia in males

Although numerous genetic and acquired factors are appreciated as risk factors for venous thromboembolism (VTE) [1,2], only recently have male gender [3,4], dyslipoproteinemia [5], and silent atherosclerotic vascular disease [6] been linked to VTE. We recently found that high-density lipoprotein (HDL) deficiency is a key feature of a pattern of dyslipoproteinemia that is associated with VTE in males, and we found that the common TaqI B1 variation in the cholesteryl ester transfer protein (CETP) gene is significantly linked to VTE [5]. However, the TaqI B1/B2 single nucleotide polymorphism (SNP) itself is unlikely to affect directly CETP activity, but it is linked to nonsynonymous CETP SNPs Ala373Pro and Arg451Gln [7–9]. Here, we demonstrate that these two CETP variations are associated with VTE and low plasma HDL levels in males.

The role of phospholipid transfer protein and cholesteryl ester transfer protein in reverse cholesterol transport

In atherosclerosis, cholesterol accumulates in the vessel wall, mainly in the form of modified low-density lipoprotein (LDL). Macrophages of the vessel wall scavenge cholesterol, which leads to formation of lipid-laden foam cells. High plasma levels of high-density lipoprotein (HDL) protect against atherosclerosis, as HDL particles can remove peripheral cholesterol and transport it to the liver for excretion in a process called reverse cholesterol transport (RCT). Phospholipid transfer protein (PLTP) remodels HDL particles in the circulation, generating prebeta-HDL and large fused HDL particles. In addition, PLTP maintains plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. Most of the cholesteryl ester transfer protein (CETP) in plasma is bound to HDL particles and CETP is also involved in the remodeling of HDL particles. CETP enhances the heteroexchange of cholesteryl esters in HDL particles for triglycerides in LDL and very low-density lipoprotein (VLDL). The aim of this thesis project was to study the importance of endogenous PLTP in the removal of cholesterol from macrophage foam cells by using macrophages derived from PLTP-deficient mice, determine the effect of macrophage-derived PLTP on the development of atherosclerosis by using bone marrow transplantation, and clarify the role of the two forms of PLTP, active and inactive, in the removal of cholesterol from the foam cells. In addition, the ability of CETP to protect HDL against the action of chymase was studied. Finally, cholesterol efflux potential of sera obtained from the study subjects was compared. The absence of PLTP in macrophages derived from PLTP-deficient mice decreased cholesterol efflux mediated by ATP-binding cassette transporter A1. The bone marrow transplantation studies showed that selective deficiency of PLTP in macrophages decreased the size of atherosclerotic lesions and caused major changes in serum lipoprotein levels. It was further demonstrated that the active form of PLTP can enhance cholesterol efflux from macrophage foam cells through generation of prebeta-HDL and large fused HDL particles enriched with apoE and phospholipids. Also CETP may enhance the RCT process, as association of CETP with reconstituted HDL particles prevented chymase-dependent proteolysis of these particles and preserved their cholesterol efflux potential. Finally, serum from high-HDL subjects promoted more efficient cholesterol efflux than did serum derived from low-HDL subjects which was most probably due to differences in the distribution of HDL subpopulations in low-HDL and high-HDL subjects. These studies described in this thesis contribute to the understanding of the PLTP/CETP-associated mechanisms underlying RCT.

Inhibitors of cholesteryl ester transfer protein - a step forward in the treatment of coronary artery disease?

Although the LDL cholesterol-lowering statins have reduced the mortality and morbidity associated with coronary artery disease (CAD), considerable mortality and morbidity remains. Increasing HDL cholesterol levels is associated with reduced CAD mortality and morbidity. In healthy subjects with mild dyslipidemia, treatment with JTT-705 decreased cholesteryl ester transfer protein (CETP) activity, increased HDL cholesterol and decreased LDL cholesterol. Similarly, another CETP inhibitor, torcetrapib, has recently been shown to increase HDL cholesterol by 46%, decrease LDL cholesterol by 8% and have no effect on triglycerides in subjects with HDL cholesterol levels below 1.0 mmol/l. Increasing HDL cholesterol with inhibitors of CETP represents a new approach to dyslipidemia that requires further investigation, especially in patients with CAD.

Interrelations between Dry Eye Syndrome and Tear Fluid Phospholipid Transfer Protein

The simplified model of human tear fluid (TF) is a three-layered structure composed of a homogenous gel-like layer of hydrated mucins, an aqueous phase, and a lipid-rich outermost layer found in the tear-air interface. It is assumed that amphiphilic phospholipids are found adjacent to the aqueous-mucin layer and externally to this a layer composed of non-polar lipids face the tear-air interface. The lipid layer prevents evaporation of the TF and protects the eye, but excess accumulation of lipids may lead to drying of the corneal epithelium. Thus the lipid layer must be controlled and maintained by some molecular mechanisms. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. The aim of this thesis was to investigate the presence and molecular mechanisms of lipid transfer proteins in human TF. The purpose was also to study the role of these proteins in the development of dry eye syndrome (DES). The presence of TF PLTP and CETP was studied by western blotting and mass spectrometry. The concentration of these proteins was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. To study the molecular mechanisms involved in PLTP mediated lipid transfer Langmuir monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) was used. Ocular tissue samples were stained with monoclonal antibodies against PLTP to study the secretion route of PLTP. Heparin-Sepharose affinity chromatography was used for PLTP pull-down experiments and co-eluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. To study whether PLTP plays any functional role in TF PLTP-deficient mice were examined. The activity of PLTP was also studied in dry eye patients. PLTP is a component of normal human TF, whereas CETP is not. TF PLTP concentration was about 2-fold higher than that in human plasma. Inactivation of PLTP by heat treatment or immunoinhibition abolished the phospholipid transfer activity in tear fluid. PLTP was found to be secreted from lacrimal glands. PLTP seems to be surface active and is capable of accepting lipid molecules without the presence of lipid-protein complexes. The active movement of radioactively labeled lipids and high activity form of PLTP to acceptor particles suggested a shuttle model of PLTP-mediated lipid transfer. In this model, PLTP physically transports lipids between the donor and acceptor. Protein-protein interaction assays revealed ocular mucins as PLTP interaction partners in TF. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression was demonstrated. Increased tear fluid PLTP activity was observed among human DES patients. These results together suggest a scavenger property of TF PLTP: if the corneal epithelium is contaminated by hydrophobic material, PLTP could remove them and transport them to the superficial layer of the TF or, alternatively, transport them through the naso-lacrimal duct. Thus, PLTP might play an integral role in tear lipid trafficking and in the protection of the corneal epithelium. The increased PLTP activity in human DES patients suggests an ocular surface protective role for this lipid transfer protein.

Glycosylation of low-density lipoprotein enhances cholesteryl ester synthesis in human monocyte-derived macrophages

Glucose can react with the lysine residues of low-density lipoproteins (LDLs) and convert the lipoprotein to a form with a receptor-mediated uptake by cultured cells that is impaired. However, in contrast to other modified lipoproteins taken up by both murine and human macrophages via the scavenger-receptor pathway that may induce the formation of foam cells, glycosylated LDL is not recognized by murine macrophages, and thus far, it has not been shown to lead to marked intracellular accumulation of cholesterol in human macrophages. This study illustrates that glycosylated LDL incubated with human monocyte-derived macrophages, at a concentration of 100 micrograms LDL/ml medium, stimulates significantly more cholesteryl ester (CE) synthesis than does control LDL (10.65 +/- 1.5 vs. 4.8 +/- 0.13 nmol.mg-1 cell protein.20 h-1; P less than .05). At LDL concentrations similar to those of plasma, the rate of CE synthesis in macrophages incubated with glycosylated LDL is more markedly enhanced than that observed in cells incubated with control LDL (3-fold increase). The marked stimulation of CE synthesis in human macrophages exposed to glycosylated LDL is paralleled by a significant increase in CE accumulation in these cells (P less than .001). The increase in CE synthesis and accumulation seem to be mediated by an increase in the degradation of glycosylated LDL by human macrophages. Glycosylated LDL enters the macrophages and is degraded by the classic LDL-receptor pathway in slightly smaller amounts than control LDL, but its degradation by pathways other than the classic LDL receptor or scavenger receptor is markedly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of cholesteryl ester synthesis in human monocyte-derived macrophages by low-density lipoproteins from type 1 (insulin-dependent) diabetic patients:the influence of non-enzymatic glycosylation of low-density lipoproteins

Diabetes mellitus is an independent risk factor in the development of atherosclerosis. In this study we aimed to demonstrate whether there is an abnormal interaction between low-density lipoproteins from diabetic patients and human macrophages. We measured cholesteryl ester synthesis and cholesteryl ester accumulation in human monocyte-derived macrophages (obtained from non-diabetic donors) incubated with low density lipoproteins from Type 1 (insulin-dependent) diabetic patients in good or fair glycaemic control. Low density lipoproteins from the diabetic patients stimulated more cholesteryl ester synthesis than low density lipoproteins from non-diabetic control subjects (7.19 +/- 1.19 vs 6.11 +/- 0.94 nmol/mg cell protein/20 h, mean +/- SEM, p less than 0.05). The stimulation of cholesteryl ester synthesis by low density lipoproteins isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (p less than 0.02). There were no significant differences in the lipid composition of low density lipoproteins between the diabetic and control groups. Non-enzymatic glycosylation of low density lipoproteins was higher in the diabetic group (p less than 0.01) and correlated significantly with cholesteryl ester synthesis (r = 0.58). Similarly, low-density lipoproteins obtained from non-diabetic subjects and glycosylated in vitro stimulated more cholesteryl ester synthesis in macrophages than control low density lipoproteins. The increase in cholesteryl ester synthesis and accumulation by cells exposed to low density lipoproteins from diabetic patients seems to be mediated by an increased uptake of these lipoproteins by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesteryl ester storage disease. Report of a case.

Cholesteryl ester storage disease (CESD) is a rare disorder of familial incidence characterized by the accumulation of cholesteryl ester and triglycerides in the liver, intestine and bone marrow. Until now only 21 cases have been reported in the literature. We present a 9 months old girl presenting with increased abdominal girth. She had normal liver function tests and increased cholesterol and triglycerides serum levels. The liver biopsy showed many cholesterol cristals seen as needle shaped cristals under polarized light. This is the youngest patient being diagnosed clinically in the literature.

Lipid Transfer to HDL is Higher in Marathon Runners than in Sedentary Subjects, but is Acutely Inhibited During the Run

Although exercise increases HDL-cholesterol, exercise-induced changes in HDL metabolism have been little explored. Lipid transfer to HDL is essential for HDL's role in reverse cholesterol transport. We investigated the effects of acute exhaustive exercise on lipid transfer to HDL. We compared plasma lipid, apolipoprotein and cytokine levels and in vitro transfer of four lipids from a radioactively labeled lipid donor nanoemulsion to HDL in sedentary individuals (n = 28) and in marathon runners (n = 14) at baseline, immediately after and 72 h after a marathon. While HDL-cholesterol concentrations and apo A1 levels were higher in marathon runners, LDL-cholesterol, apo B and triacylglycerol levels were similar in both groups. Transfers of non-esterified cholesterol [6.8 (5.7-7.2) vs. 5.2 (4.5-6), p = 0.001], phospholipids [21.7 (20.4-22.2) vs. 8.2 (7.7-8.9), p = 0.0001] and triacylglycerol [3.7 (3.1-4) vs. 1.3 (0.8-1.7), p = 0.0001] were higher in marathon runners, but esterified-cholesterol transfer was similar. Immediately after the marathon, LDL- and HDL-cholesterol concentrations and apo A1 levels were unchanged, but apo B and triacylglycerol levels increased. Lipid transfer of non-esterified cholesterol [6.8 (5.7-7.2) vs. 5.8 (4.9-6.6), p = 0.0001], phospholipids [21.7 (20.4-22.2) vs. 19.1 (18.6-19.3), p = 0.0001], esterified-cholesterol [3.2 (2.2-3.8) vs. 2.3 (2-2.9), p = 0.02] and triacylglycerol [3.7 (3.1-4) vs. 2.6 (2.1-2.8), p = 0.0001] to HDL were all reduced immediately after the marathon but returned to baseline 72 h later. Running a marathon increased IL-6 and TNF-alpha levels, but after 72 h these values returned to baseline. Lipid transfer, except esterified-cholesterol transfer, was higher in marathon runners than in sedentary individuals, but the marathon itself acutely inhibited lipid transfer. In light of these novel observations, further study is required to clarify how these metabolic changes can influence HDL composition and anti-atherogenic function.

Plasma lipases and lipid transfer proteins increase phospholipid but not free cholesterol transfer from lipid emulsion to high density lipoproteins.

Abstract Background Plasma lipases and lipid transfer proteins are involved in the generation and speciation of high density lipoproteins. In this study we have examined the influence of plasma lipases and lipid transfer protein activities on the transfer of free cholesterol (FC) and phospholipids (PL) from lipid emulsion to human, rat and mouse lipoproteins. The effect of the lipases was verified by incubation of labeled (3H-FC,14C-PL) triglyceride rich emulsion with human plasma (control, post-heparin and post-heparin plus lipase inhibitor), rat plasma (control and post-heparin) and by the injection of the labeled lipid emulsion into control and heparinized functionally hepatectomized rats. Results In vitro, the lipase enriched plasma stimulated significantly the transfer of 14C-PL from emulsion to high density lipoprotein (p<0.001) but did not modify the transfer of 3H-FC. In hepatectomized rats, heparin stimulation of intravascular lipolysis increased the plasma removal of 14C-PL and the amount of 14C-PL found in the low density lipoprotein density fraction but not in the high density lipoprotein density fraction. The in vitro and in vivo experiments showed that free cholesterol and phospholipids were transferred from lipid emulsion to plasma lipoproteins independently from each other. The incubation of human plasma, control and control plus monoclonal antibody anti-cholesteryl ester transfer protein (CETP), with 14C-PL emulsion showed that CETP increases 14C-PL transfer to human HDL, since its partial inhibition by the anti-CETP antibody reduced significantly the 14C-PL transfer (p<0.05). However, comparing the nontransgenic (no CETP activity) with the CETP transgenic mouse plasma, no effect of CETP on the 14C-PL distribution in mice lipoproteins was observed. Conclusions It is concluded that: 1-intravascular lipases stimulate phospholipid transfer protein mediated phospholipid transfer, but not free cholesterol, from triglyceride rich particles to human high density lipoproteins and rat low density lipoproteins and high density lipoproteins; 2-free cholesterol and phospholipids are transferred from triglyceride rich particles to plasma lipoproteins by distinct mechanisms, and 3 - CETP also contributes to phospholipid transfer activity in human plasma but not in transgenic mice plasma, a species which has high levels of the specific phospholipid transfer protein activity.