Teus van Gent

Human Plasma Phospholipid Transfer Protein Increases the Antiatherogenic Potential of High Density Lipoproteins in Transgenic Mice

Plasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress human PLTP were generated. Compared with wild-type mice, these mice show a 2.5- to 4.5-fold increase in PLTP activity in plasma. This results in a 30% to 40% decrease of plasma levels of HDL cholesterol. Incubation of plasma from transgenic animals at 37 degrees C reveals a 2- to 3-fold increase in the formation of pre-beta-HDL compared with plasma from wild-type mice. Although pre-beta-HDL is normally a minor subfraction of HDL, it is known to be a very efficient acceptor of peripheral cell cholesterol and a key mediator in reverse cholesterol transport. Further experiments show that plasma from transgenic animals is much more efficient in preventing the accumulation of intracellular cholesterol in macrophages than plasma from wild-type mice, despite lower total HDL concentrations. It is concluded that PLTP can act as an antiatherogenic factor preventing cellular cholesterol overload by generation of pre-beta-HDL.

Increased risk of atherosclerosis by elevated plasma levels of phospholipid transfer protein

Plasma phospholipid transfer protein (PLTP) is thought to be involved in the remodeling of high density lipoproteins (HDL), which are atheroprotective. It is also involved in the metabolism of very low density lipoproteins (VLDL). Hence, PLTP is thought to be an important factor in lipoprotein metabolism and the development of atherosclerosis. We have overexpressed PLTP in mice heterozygous for the low density lipoprotein (LDL) receptor, a model for atherosclerosis. We show that increased PLTP activity results in a dose-dependent decrease in HDL, and a moderate stimulation of VLDL secretion (≤1.5-fold). The mice were given a high fat, high cholesterol diet, which resulted in hypercholesterolemia in all animals. HDL concentrations were dramatically reduced in PLTP-overexpressing animals when compared with LDL receptor controls, whereas VLDL + LDL cholesterol levels were identical. Susceptibility to atherosclerosis was increased in a PLTP dose-responsive manner. We conclude that PLTP increases susceptibility to atherosclerosis by lowering HDL concentrations, and therefore we suggest that an increase in PLTP is a novel, long term risk factor for atherosclerosis in humans.

Dietary trans fatty acids increase serum cholesterylester transfer protein activity in man

The average diet may provide some 8-10 g/day of unsaturated fatty acids with a trans double bond. Previous studies showed that dietary trans fatty acids may simultaneously raise low-density lipoprotein (LDL) cholesterol and reduce high-density lipoprotein (HDL) cholesterol. Human plasma contains a protein (CETP) which transfers cholesterylesters from HDL to lipoproteins of lower density. We hypothesized that CETP could play a role in the effect of trans fatty acids on lipoproteins and measured the activity levels of CETP in serum samples from a 9-week study in which 55 volunteers were fed three controlled diets with different fatty acid profiles. Mean activity was 114 (% of reference serum) after consumption of a high trans fatty acid diet, as opposed to 96 after linoleic acid and 97 after stearic acid (P < 0.02). We conclude that the increased activity of CETP may contribute to the rise in LDL cholesterol and the fall in HDL cholesterol seen on diets with high contents of trans fatty acids.

Functional Expression of Endothelial Nitric Oxide Synthase Fused to Green Fluorescent Protein in Transgenic Mice

The activity of endothelial nitric oxide synthase (eNOS) is subject to complex transcriptional and post-translational regulation including the association with several proteins and variations in subcellular distribution. In the present study we describe a transgenic mouse model expressing eNOS fused to green fluorescent protein (GFP), which allows the study of localization and regulation of eNOS expression. We tested the functionality of eNOS in the eNOS-GFP mice. Expression of eNOS was restricted to the endothelial lining of blood vessels in various tissues tested, without appreciable expression in non-endothelial cells. Activity of the enzyme was confirmed by assaying the conversion of L-arginine to L-citrulline. NO production in isolated vessels was increased in transgenic mice when compared to non-transgenic control animals (4.88 +/- 0.59 and 2.48 +/- 0.47 micro mol/L NO, respectively, P < 0.005). Both the mean aortic pressure and the pulmonary artery pressure were reduced in eNOS-GFP mice (both approximately 30%, P < 0.05). Plasma cholesterol levels were also slightly reduced ( approximately 20%, P < 0.05). In conclusion, eNOS-GFP mice express functional eNOS and provide a unique model to study regulation of eNOS activity or eNOS-mediated vascular events, including response to ischemia, response to differences in shear stress, angiogenesis and vasculogenesis, and to study the subcellular distribution in relation with functional responses to these events.

Changes in postprandial lipoproteins of low and high density caused by moderate alcohol consumption with dinner

We measured the effects of consumption of moderate amounts of beer, wine or spirits with evening dinner on plasma LDL and HDL levels as well as composition in 11 healthy middle-aged men. Forty grams of alcohol were consumed daily with dinner for a period of 3 weeks. Mineral water was used as a negative control. Dinner was served at 6 pm and blood samples were obtained at 1 h before and 3, 5, 9, and 13 h after the start of the meal. No differences were detected between the effects of the different alcohol-containing beverages. Plasma levels of triglycerides (TG), measured 1 h before dinner were very variable and higher than fasting values (means of 2.2 and 1.5 mM, respectively). Daily consumption of 40 g of alcohol with dinner resulted in increased postprandial plasma TG levels and decreased low density lipoprotein (LDL) cholesterol concentrations. These effects were transient and observed at 11 pm (TG) and 9 pm and 11 pm (LDL). In contrast, high density lipoproteins (HDL) were raised by alcohol intake at all time points analysed. HDL composition was changed by alcohol consumption, resulting in a raised HDL-cholesterol/apo A-I ratio at 5 pm and 9 pm. The observed alcohol-dependent effects on plasma HDL and LDL during the postprandial phase are considered anti-atherogenic and may contribute to the observed protection against coronary heart disease by moderate alcohol consumption.

Evaluation of phospholipid transfer protein and cholesteryl ester transfer protein as contributors to the generation of pre beta-high-density lipoproteins.

High-density lipoproteins (HDLs) are considered anti-atherogenic because they mediate peripheral cell cholesterol transport to the liver for excretion and degradation. An important step in this reverse cholesterol-transport pathway is the uptake of cellular cholesterol by a specific subclass of small, lipid-poor apolipoprotein A-I particles designated pre beta-HDL. The two lipid-transfer proteins present in human plasma, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), have both been implicated in the formation of pre beta-HDL. In order to investigate the relative contribution of each of these proteins, we used transgenic mouse models. Comparisons were made between human CETP transgenic mice (huCETPtg), human PLTP transgenic mice (huPLTPtg) and mice transgenic for both lipid-transfer proteins (huCETPtg/huPLTPtg). These animals showed elevated plasma levels of CETP activity, PLTP activity or both activities, respectively. We evaluated the generation of pre beta-HDL in mouse plasma by immunoblotting and crossed immuno-electrophoresis. Generation of pre beta-HDL was equal in huCETPtg and wild-type mice. In contrast, in huPLTPtg and huCETPtg/huPLTPtg mice, pre beta-HDL generation was 3-fold higher than in plasma from either wild-type or huCETPtg mice. Our findings demonstrate that, of the two plasma lipid-transfer proteins, PLTP rather than CETP is responsible for the generation of pre beta-HDL. These data support the hypothesis of a role for PLTP in the initial stage of reverse cholesterol transport.

Genome-wide DNA methylation profiling of non-small cell lung carcinomas

BackgroundNon-small cell lung carcinoma (NSCLC) is a complex malignancy that owing to its heterogeneity and poor prognosis poses many challenges to diagnosis, prognosis and patient treatment. DNA methylation is an important mechanism of epigenetic regulation involved in normal development and cancer. It is a very stable and specific modification and therefore in principle a very suitable marker for epigenetic phenotyping of tumors. Here we present a genome-wide DNA methylation analysis of NSCLC samples and paired lung tissues, where we combine MethylCap and next generation sequencing (MethylCap-seq) to provide comprehensive DNA methylation maps of the tumor and paired lung samples. The MethylCap-seq data were validated by bisulfite sequencing and methyl-specific polymerase chain reaction of selected regions.ResultsAnalysis of the MethylCap-seq data revealed a strong positive correlation between replicate experiments and between paired tumor/lung samples. We identified 57 differentially methylated regions (DMRs) present in all NSCLC tumors analyzed by MethylCap-seq. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples. We also identified DMRs that were specific to two of the major subtypes of NSCLC, adenocarcinomas and squamous cell carcinomas.ConclusionsCollectively, we provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC.

Saturable high affinity binding, uptake and degradation of rat plasma lipoproteins by isolated parenchymal and non-parenchymal cells from rat liver

Abstract 1. Freshly isolated rat parenchymal and non-parenchymal liver cells bind iodinated rat very low density lipoprotein (VLDL) remnants, low density lipoproteins (LDL) and high density lipoproteins (HDL) in a saturable way. The apparent K m values for the binding of VLDL remnants are 6–20-fold lower than for LDL or HDL. The binding per mg cell protein to non-parenchymal cells is 5–8-fold higher than to parenchymal cells. Competition experiments indicate that rat VLDL-remnants, LDL and HDL, but not human LDL compete for the same surface receptor. It is concluded that the source of common recognition could be apolipoprotein E and that the interaction with the receptor is also influenced by the apolipoproteins A and C. The high apparent affinity of the receptor for VLDL remnants might be the result of multiple receptor occupancy of this lipoprotein. The presence of a 5–8-fold higher concentration of the described lipoprotein receptor in non-parenchymal cells as compared to parenchymal cells explains the relatively high uptake of VLDL remnants (as compared to LDL and HDL) as well as the relative contribution of parenchymal and non-parenchymal cells to the total hepatic uptake of lipoproteins in vivo. 2. The greater part (70–80%) of the parenchymal and non-parenchymal cell-associated apolipoproteins, LDL or HDL, remains bound to the external surface of the cells, during in vitro incubation at 37°C. High-affinity degradation of apolipoprotein(s) by isolated liver cells is dependent on the specific lipoprotein. During a 3 h incubation at 37°C, 37–49% of the total cell-associated 125 I-abeled HDL is degraded. These percentages are 11–13% for 125 I-labeled LDL and 4–8% for 125 I-labeled VLDL remnants. Degradation of the different lipoproteins by non-parenchymal liver cells occurs at a 3–6-times higher rate per mg cell protein than by parenchymal cells. It is suggested that the rate-limiting step in the degradation of apolipoprotein by isolated liver cells is their transport to intracellular degradation sites.

Saturable high affinity binding of low density and high density lipoprotein by parenchymal and non-parenchymal cells from rat liver

Abstract Freshly isolated parenchymal liver cells bind both low density lipoprotein (LDL) and high density lipoprotein (HDL). With increasing concentrations of LDL and HDL the amount of cell-associated radioactivity approaches saturation and a linear double-reciprocal plot for the binding is obtained. The binding of LDL and HDL to isolated non-parenchymal liver cells is also saturable and the maximal binding of LDL and HDL per mg cell protein is 4–5 times higher than with parenchymal cells. It is suggested that the presence of a 4–5 fold higher concentration of lipoprotein receptor (for LDL and HDL) on non-parenchymal cells as compared to parenchymal cells explains the 4–5 times higher uptake of lipoproteins by the non-parenchymal liver cells, observed in vivo .

Decreased PLTP mass but elevated PLTP activity linked to insulin resistance in HTG: effects of bezafibrate therapy

Hypertriglyceridemia (HTG) is associated with insulin resistance, increased cholesteryl ester transfer (CET), and low HDL cholesterol. Phospholipid transfer protein (PLTP) may be involved in these relationships. Associations between CET, lipids, insulin resistance, CETP and PLTP activities, and PLTP mass were investigated in 18 HTG patients and 20 controls. Effects of 6 weeks of bezafibrate treatment were studied in HTG patients. HTG patients had higher serum triglycerides, insulin resistance, free fatty acid (FFA), and CET, lower levels of HDL cholesterol (−44%) and PLTP mass (−54%), and higher CETP (+20%) and PLTP activity (+48%) than controls. Bezafibrate reduced triglycerides, CET (−37%), insulin resistance (−53%), FFA (−48%), CETP activity (−12%), PLTP activity (−8%), and increased HDL cholesterol (+27%), whereas PLTP mass remained unchanged. Regression analysis showed a positive contribution of PLTP mass (P = 0.001) but not of PLTP activity to HDL cholesterol, whereas insulin resistance positively contributed to PLTP activity (P < 0.01). Bezafibrate-induced change in CET and HDL cholesterol correlated with changes in CETP activity and FFAs, but not with change in PLTP activity. Bezafibrate-induced change in PLTP activity correlated with change in FFAs (r = 0.455, P = 0.058). We propose that elevated PLTP activity in HTG is related to insulin resistance and not to increased PLTP mass. Bezafibrate-induced diminished insulin resistance is associated with a reduction of CET and PLTP activity.