Arie van Tol

Efficacy and Safety of a Novel Cholesteryl Ester Transfer Protein Inhibitor, JTT-705, in Humans A Randomized Phase II Dose-Response Study

Background—Cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins. High plasma levels of CETP are correlated with low HDL cholesterol levels, a strong risk factor for coronary artery disease. In earlier studies, JTT-705, a novel CETP inhibitor, was shown to increase plasma HDL cholesterol and to inhibit the progression of atherosclerosis in cholesterol-fed rabbits. This study describes the first results using this CETP inhibitor in humans. Methods and Results—In a randomized, double-blind, and placebo-controlled trial, we evaluated the efficacy and safety of daily treatment with 300, 600, and 900 mg JTT-705 in 198 healthy subjects with mild hyperlipidemia. Treatment with 900 mg JTT-705 for 4 weeks led to a 37% decrease in CETP activity (P <0.0001), a 34% increase in HDL cholesterol (P <0.0001), and a 7% decrease in LDL cholesterol (P =0.017), whereas levels of triglycerides, phospholipid transfer protein, and lecithin-cholesterol acyltransferase were unaffected. In line with the increase of total HDL, a rise of HDL2, HDL3, and apolipoprotein A-I was also noted. JTT-705 showed no toxicity with regard to physical examination and routine laboratory tests. Conclusions—We show that the use of the CETP inhibitor JTT-705 in humans is an effective means to raise HDL cholesterol levels with minor gastrointestinal side effects (P =0.06). Although these results hold promise, further studies are needed to investigate whether the observed increase in HDL cholesterol translates into a concomitant reduction in coronary artery disease risk.

Proatherogenic Role of Elevated CE Transfer From HDL to VLDL1 and Dense LDL in Type 2 Diabetes

Abstract —Plasma cholesteryl ester transfer protein (CETP) facilitates intravascular lipoprotein remodeling by promoting the heteroexchange of neutral lipids. To determine whether the degree of triglyceridemia may influence the CETP-mediated redistribution of HDL CE between atherogenic plasma lipoprotein particles in type 2 diabetes, we evaluated CE mass transfer from HDL to apoB-containing lipoprotein acceptors in the plasma of type 2 diabetes subjects (n=38). In parallel, we investigated the potential relationship between CE transfer and the appearance of an atherogenic dense LDL profile. The diabetic population was divided into 3 subgroups according to fasting plasma triglyceride (TG) levels: group 1 (G1), TG<100 mg/dL; group 2 (G2), 100200 mg/dL. Type 2 diabetes patients displayed an asymmetrical LDL profile in which the dense LDL subfractions predominated. Plasma levels of dense LDL subfractions were strongly positively correlated with those of plasma triglyceride (TG) (r =0.471;P =0.0003). The rate of CE mass transfer from HDL to apoB-containing lipoproteins was significantly enhanced in G3 compared with G2 or G1 (46.2±8.1, 33.6±5.3, and 28.2±2.7 &mgr;g CE transferred · h−1 · mL−1 in G3, G2, and G1, respectively;P <0.0001 G3 versus G1, P =0.0001 G2 versus G1, and P =0.02 G2 versus G3). The relative capacities of VLDL and LDL to act as acceptors of CE from HDL were distinct between type 2 diabetes subgroups. LDL particles represented the preferential CE acceptor in G1 and accounted for 74% of total CE transferred from HDL. By contrast, in G2 and G3, TG-rich lipoprotein subfractions accounted for 47% and 72% of total CE transferred from HDL, respectively. Moreover, the relative proportion of CE transferred from HDL to VLDL1 in type 2 diabetes patients increased progressively with increase in plasma TG levels. The VLDL1 subfraction accounted for 34%, 43%, and 52% of total CE transferred from HDL to TG-rich lipoproteins in patients from G1, G2, and G3, respectively. Finally, dense LDL acquired an average of 45% of total CE transferred from HDL to LDL in type 2 diabetes patients. In conclusion, CETP contributes significantly to the formation of small dense LDL particles in type 2 diabetes by a preferential CE transfer from HDL to small dense LDL, as well as through an indirect mechanism involving an enhanced CE transfer from HDL to VLDL1, the specific precursors of small dense LDL particles in plasma.

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.

Elevated plasma cholesteryl ester transfer in NIDDM: relationships with apolipoprotein B-containing lipoproteins and phospholipid transfer protein

Lecithin:cholesteryl acyl transferase (LCAT) and cholesteryl ester transfer protein (CETP) are key factors in the esterification of cholesterol and the subsequent transfer of cholesteryl ester from high density lipoproteins (HDL) towards very low and low density lipoproteins (VLDL + LDL). Phospholipid transfer protein (PLTP), lipoprotein lipase (LPL) and hepatic lipase (HL) are involved in plasma phospholipid and triglyceride metabolism and also affect HDL. Equivocal changes in plasma cholesteryl ester transfer have been reported in non-insulin-dependent diabetes mellitus (NIDDM). In 16 NIDDM men with plasma triglycerides < or = 4.5 mmol/l and cholesterol < or = 8.0 mmol/l. plasma cholesteryl ester transfer (CET), cholesterol esterification rate, LCAT and PLTP activity levels were higher (P < 0.05 to P < 0.02) in conjunction with higher plasma triglycerides (P < 0.01) and lower HDL cholesterol and cholesteryl ester levels (P < 0.05) compared to 16 matched healthy men. Multiple stepwise regression analysis demonstrated that CET was positively related to VLDL + LDL cholesterol (P < 0.001), triglycerides (P = 0.001), PLTP activity (P = 0.007) and CETP activity (P = 0.008, multiple r = 0.94). NIDDM had no effect on CET, independently from these parameters. HDL cholesteryl ester was negatively related to CET (P= 0.017), HL activity (P = 0.033) and NIDDM (P = 0.047) and positively to LCAT activity levels (P = 0.034, multiple r = 0.68). It is concluded that the elevated CET in plasma from NIDDM patients is associated with higher plasma triglycerides and PLTP activity levels. Furthermore, our data suggest that in normo- and moderately dyslipidaemic subjects PLTP and CETP activity levels per se may influence the rate of cholesteryl ester transfer in plasma. Plasma cholesteryl ester transfer appears to be a determinant of HDL cholesteryl ester, but other factors are likely to contribute to lower HDL cholesteryl ester levels in NIDDM.

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.

Dose-dependent action of atorvastatin in type IIB hyperlipidemia: preferential and progressive reduction of atherogenic apoB- containing lipoprotein subclasses (VLDL-2, IDL, small dense LDL) and stimulation of cellular cholesterol efflux

Type IIB hyperlipidemia is associated with premature vascular disease, an atherogenic lipoprotein phenotype characterised by elevated levels of triglyceride-rich VLDL and small dense LDL, together with subnormal levels of HDL. The dose-dependent and independent effects of a potent HMGCoA reductase inhibitor, Atorvastatin, at daily doses of 10 and 40 mg, were evaluated on triglyceride-rich lipoprotein subclasses (VLDL-1, VLDL-2 and IDL), on the major LDL subclasses (light LDL, LDL-1+LDL-2, D: 1.019-1.029 g/ml; intermediate LDL, LDL-3, D: 1.029-1.039 g/ml and small dense LDL, LDL-4+LDL+5, D: 1.039-1.063 g/ml), on CETP-mediated cholesteryl ester transfer from HDL to apoB-containing lipoproteins, on phospholipid transfer protein activity and on plasma-mediated cellular cholesterol efflux in patients (n=10) displaying type IIB hyperlipidemia. Plasma concentrations of triglyceride-rich lipoprotein subclasses (TRL: VLDL-1, Sf 60-400; VLDL-2, Sf 20-60 and IDL, Sf 12-20) and of LDL (D: 1.019-1.063 g/ml) were markedly diminished after 6 weeks of statin treatment at 10 mg per day (-31 and -36%, respectively; P<0.002) and by 42 and 51%, respectively at the 40 mg per day dose. Increasing doses of atorvastatin progressively normalised both the quantitative and qualitative features of the LDL subclass profile, in which dense LDL predominated at baseline. Indeed, dense LDL levels were reduced by up to 57% at the 40-mg dose, leading to a shift in the peak of the density profile towards larger, buoyant LDL particles typical of normolipidemic subjects. In addition, marked reduction in numbers of apoB100-containing particle acceptors led to a 30% decrease (P<0.02) in CETP-mediated CE transfer from HDL. Finally, a significant dose-dependent statin-mediated elevation (+15% at 10 mg; P=0.0003 and +35% at 40 mg; P<0.0001 compared to baseline) in the capacity of plasma from type IIB subjects to mediate free cholesterol efflux from Fu5AH hepatoma cells was observed. Moreover, atorvastatin (40 mg per day) significantly increased plasma apoAI levels (+24%; P<0.05), thereby suggesting that this statin enhances production of apoAI and with it, formation of nascent pre-beta HDL particles. Plasma PLTP activity was not affected by either dose of atorvastatin. We conclude that increasing the dose of atorvastatin leads to dose-dependent, preferential and progressive reduction in particle numbers of atherogenic VLDL-2, IDL and dense LDL, and concomitantly, to enhanced cellular cholesterol efflux in type IIB dyslipidemia, thereby diminishing the atherosclerotic burden in subjects characterised by high cardiovascular risk.

Kinetics of HDL cholesterol and paraoxonase activity in moderate alcohol consumers.

BACKGROUND The inverse association between moderate drinking and coronary heart disease mortality is well established. This study was performed to investigate the kinetics of the alcohol-induced increases in apo A-1, HDL cholesterol, and paraoxonase (PON) activity, as well as to study whether the alcohol-induced increases in PON activity differ within different PON polymorphisms, and to investigate whether moderate alcohol consumption has similar effects on the outcome measures in postmenopausal women as in middle-aged men. METHODS In a randomized, diet-controlled, crossover study, 10 middle-aged men and 9 postmenopausal women, all apparently healthy, nonsmoking, and moderate alcohol drinkers, consumed beer or no-alcohol beer (control) with evening dinner during two successive periods of 3 weeks. During the beer period, alcohol intake equaled 40 and 30 g/day for men and women, respectively. The total diet was supplied to the subjects and had essentially the same composition during these 6 weeks. Before each treatment was a 1-week washout period, in which the subjects were not allowed to drink alcoholic beverages. RESULTS Moderate alcohol consumption significantly increased serum apo A-I level after 5 days (3.7%, p < 0.05); after 10 days, serum HDL cholesterol level was increased (6.8%, p < 0.001), and after 15 days serum PON activity was increased (3.7%, p < 0.05), all compared with no alcohol consumption. Gene polymorphisms did not modulate the alcohol effect on PON. CONCLUSIONS Serum apo A-I, HDL cholesterol, and PON activity were significantly increased during 3 weeks of moderate alcohol consumption as compared with no alcohol consumption. Moreover, the results suggest that there is a sequence in induction of these parameters. After an increase in apo A-I, HDL cholesterol is increased followed by an increase in PON activity. Increased serum HDL cholesterol level and PON activity may be a mechanism of action not only in healthy middle-aged men but also in postmenopausal women, underlying the reduced coronary heart disease risk in moderate drinkers.

Hepatic lipase and lipoprotein lipase are not major determinants of the low density lipoprotein subclass pattern in human subjects with coronary heart disease

The influence of hepatic lipase (HL) and lipoprotein lipase (LPL) activity on the low density lipoprotein (LDL) subclass pattern was studied in a population of males with coronary heart disease and without severe hypercholesterolemia. LDL subclass patterns, lipases and plasma lipoproteins were determined in 326 patients. In part of the study population, fasting insulin and glucose levels were also determined. The LDL subclass pattern was determined by gradient gel electrophoresis (GGE) and classified according to Austin et al. (J. Am. Med. Assoc. 260 (1988) 1917 (predominantly large LDL = A-pattern, predominantly small LDL = B-pattern). An LDL subclass A-pattern was exhibited by 199 subjects; 108 exhibited a B-pattern. In 19 subjects no distinctive A- or B-pattern was present (A/B-pattern). Hepatic and lipoprotein activities differed significantly between patients with the A- or B-pattern. The median hepatic lipase activity was lower (384 vs. 417 mU/ml, P = 0.006), and the lipoprotein lipase activity higher (122 vs. 101 mU/ml, P = 0.001) in the A-pattern subjects than in the B-pattern subjects. In subjects with the A/B pattern the lipase activities were intermediate between the values in the A- and B-pattern subjects (HL 408 +/- 87 mU/ml, LPL 115 +/- 55 mU/ml). Plasma triglyceride, very low density lipoprotein (VLDL)-triglyceride, intermediate density lipoprotein (IDL)-triglyceride and LDL-triglyceride were higher in the patients with a B-pattern (+84%, +171%, +10% and +16%, respectively). Total plasma cholesterol was not different between A- and B-pattern subjects. VLDL- and IDL-cholesterol were higher in the B-pattern group (+174% and +66%, respectively), while LDL- and HDL-cholesterol were higher in the A-pattern group (+2 and +24%, respectively). In univariate analysis HL, LPL, plasma (and VLDL) triglyceride, HDL-cholesterol and IDL-cholesterol were each significantly associated with the LDL subclass pattern. In multivariate analysis plasma triglyceride (or VLDL-triglyceride) and HDL-cholesterol appeared to be independently associated with the LDL subclass pattern. No additional discriminative value of HL or LPL was found. Similar results were obtained if the patients with or without beta blocker were evaluated separately. An estimate of insulin resistance (EIR), calculated from plasma insulin and glucose in part of the study population (n = 145), was significantly higher in the subjects with a B-pattern than in those with an A-pattern (3.12 vs. 2.00, P < 0.003). EIR correlated positively with plasma triglyceride (P < 0.0001), but not with HL or LPL.(ABSTRACT TRUNCATED AT 400 WORDS)

Moderate alcohol consumption: effects on lipids and cardiovascular disease risk.

Light to moderate alcohol consumption is associated with a reduced risk of coronary heart disease, as well as ischaemic stroke and possibly type 2 diabetes. Epidemiological and physiological data are in favour of a causal relationship. Proposed protective mechanisms include the stimulation of HDL-mediated processes such as reverse cholesterol transport and antioxidative effects. More well-controlled studies are needed to provide a complete understanding of the complexity of the underlying physiological mechanisms.

Genetic Variation at the Phospholipid Transfer Protein Locus Affects Its Activity and High-Density Lipoprotein Size and Is a Novel Marker of Cardiovascular Disease Susceptibility

Background— In contrast to clear associations between variants in genes participating in low-density lipoprotein metabolism and cardiovascular disease risk, such associations for high-density lipoprotein (HDL)–related genes are not well supported by recent large studies. We aimed to determine whether genetic variants at the locus encoding phospholipid transfer protein (PLTP), a protein involved in HDL remodeling, underlie altered PLTP activity, HDL particle concentration and size, and cardiovascular disease risk. Methods and Results— We assessed associations between 6 PLTP tagging single nucleotide polymorphisms and PLTP activity in 2 studies (combined n=384) and identified 2 variants that show reproducible associations with altered plasma PLTP activity. A gene score based on these variants is associated with lower hepatic PLTP transcription (P=3.2×10−18) in a third study (n=957) and with an increased number of HDL particles of smaller size (P=3.4×10−17) in a fourth study (n=3375). In a combination of 5 cardiovascular disease case-control studies (n=4658 cases and 11 459 controls), a higher gene score was associated with a lower cardiovascular disease risk (per-allele odds ratio, 0.94; 95% confidence interval, 0.90 to 0.98; P=1.2×10−3; odds ratio for highest versus lowest gene score, 0.69; 95% confidence interval, 0.55 to 0.86; P=1.0×10−3). Conclusions— A gene score based on 2 PLTP single nucleotide polymorphisms is associated with lower PLTP transcription and activity, an increased number of HDL particles, smaller HDL size, and decreased risk of cardiovascular disease. These findings indicate that PLTP is a proatherogenic entity and suggest that modulation of specific elements of HDL metabolism may offer cardiovascular benefit.