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Probucol markedly reduces HDL phospholipids and elevated preβ1-HDL without delayed conversion into α-migrating HDL: Putative role of angiopoietin-like protein 3 in probucol-induced HDL remodeling
Atherosclerosis 200 (2008) 329–335
Probucol markedly reduces HDL phospholipids and elevated pre1-HDL without delayed conversion into ␣-migrating HDL: Putative role of angiopoietin-like protein 3 in probucol-induced HDL remodeling Takashi Miida a,∗ , Utako Seino b , Osamu Miyazaki c , Osamu Hanyu d , Satoshi Hirayama d , Toshikazu Saito e , Yuichi Ishikawa e , Suguru Akamatsu e , Toshimitsu Nakano e , Katsuyuki Nakajima e , Mitsuyo Okazaki f , Masahiko Okada b a
e
Department of Clinical Laboratory Medicine, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan b Division of Clinical Preventive Medicine, Department of Community Preventive Medicine, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi 1-757, Chuo-ku, Niigata, Niigata 951-8510, Japan c Diagnostics Research Laboratories, Daiichi Pure Chemicals, Koyodai 3-3-1, Ryugasaki, Ibaraki 301-0852, Japan d Division of Endocrinology and Metabolism, Department of Homeostatic Regulation and Developments, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi 1-757, Chuo-ku, Niigata, Niigata 951-8510, Japan Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Shinagawa Grand Central Tower, Konan 2-16-4, Minato-Ku, Tokyo 108-8242, Japan f Laboratory of Chemistry, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Kohnodai 2-8-30, Ichikawa, Chiba 272-0827, Japan Received 16 November 2007; accepted 27 December 2007 Available online 14 February 2008
Abstract Probucol is a unique hypolipidemic agent that increases cholesteryl ester transfer protein (CETP) activity. Enhanced CETP-mediated conversion of high-density lipoprotein (HDL) partly explains the probucol-induced decrease in HDL cholesterol and increase in plasma pre1-HDL (native lipid-poor HDL) concentrations. However, HDL cholesterol is reduced in patients that are completely deficient in CETP. Angiopoietin-like protein 3 (ANGPTL3) is an endogenous suppressor of endothelial lipase that promotes the hydrolysis of HDL phospholipids and may generate pre1-HDL. To determine whether probucol decreases ANGPTL3 and HDL phospholipids while increasing pre1-HDL, we measured these parameters before and after a 4-week probucol treatment in 39 hypercholesterolemic patients and age- and sex-matched controls. The median ANGPTL3 had decreased from 143 to 113 g/L by week 4 (p < 0.05). High-performance liquid chromatography revealed that probucol decreased the phospholipid content of very large (13.5–15 nm) and large (12.1 nm) HDL particles predominantly by 65% (p < 0.01) and 53% (p < 0.001), respectively. The change in ANGPTL3, but not CETP mass, was positively correlated with that in large HDL phospholipids (r = 0.455, p < 0.05). The absolute and relative concentrations of pre1-HDL increased by 14% (p < 0.01) and 60% (p < 0.001), respectively. The conversion rate of preβ1-HDL into α-migrating HDL by lecithin-cholesterol acyltransferase did not change significantly. In conclusion, probucol decreases plasma ANGPTL3 and HDL phospholipids while increasing pre1-HDL. We speculate that probucol induces HDL remodeling via an endothelial lipase-mediated pathway. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: ABCA1; Endothelial lipase; Inflammation; LCAT; Reverse cholesterol transport
1. Introduction
∗
Corresponding author. Tel.: +81-3-5802-1104; fax: +81-3-5684-1609. E-mail address: [email protected] (T. Miida).
0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.12.031
Numerous epidemiological studies clearly show that low high-density lipoprotein cholesterol (HDL-C) is a significant predictor of cardiovascular disease [1]. This favorable effect of HDL is related to its ability to remove excess
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cholesterol from peripheral tissues and transport it to the liver for further metabolism (reverse cholesterol transport; RCT). HDL consists of distinct particles that have various compositions and physiological functions; these can be classified into several HDL subfractions using different methods. Pre1-HDL is a distinct lipid-poor HDL Lp-AI subfraction (the lipoprotein contains apolipoprotein AI (apoAI), but no apoAII) [2]. Previously, we showed that pre1-HDL accelerates the cholesterol efflux from cultured fibroblasts in a dose-dependent manner [3]. Several lines of evidence suggest that plasma pre1-HDL is at least partly generated by the action of lipase [4,5]. Probucol is a mild cholesterol-lowering agent that is a strong antioxidant. Probucol treatment increases the pre1HDL concentration [6]. Because probucol increases both CETP mass and activity [6–8], the CETP-dependent conversion of large spherical HDL is most likely to contribute to the increase in pre1-HDL concentration in probucol-treated patients [6]. However, probucol reportedly even decreases HDL-C and HDL particle size in patients completely deficient in CETP [9], suggesting that it also affects HDL metabolism via a CETP-independent mechanism. Angiopoietin-like protein 3 (ANGPTL3) is a novel endogenous inhibitor of lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL), and endothelial lipase (EL) [10,11]. EL is a novel member of the lipase family that hydrolyzes HDL phospholipids (PL) [11]. During this process, pre1-HDL is likely generated from the surface remnants of hydrolyzed HDL. Therefore, probucol may promote HDL remodeling via the ANGPTL3-EL pathway. We examined whether probucol decreases ANGPTL3 and HDL-PL while increasing pre1-HDL in patients with hypercholesterolemia. To measure these parameters, we used our own enzyme-linked immunoassays for ANGPTL3 and pre1-HDL and a high-performance liquid chromatography (HPLC) system with analytical software for HDL phospholipids.
2. Materials and methods 2.1. Patients and study design We recruited study subjects from consecutive outpatients with hypercholesterolemia [total cholesterol (TC) concentration >5.69 mmol/L; 220 mg/dL] [12] at our institution. No patient had received a lipid-lowering agent previously. Routine laboratory tests revealed normal liver and kidney function. We excluded patients with a history of coronary heart disease or clinical symptoms of acute or chronic infection such as cough, sputum, and fever in the preceding 2 months. Thus, 39 patients aged 38–75 years were enrolled in the study. Age- and sex-matched control subjects were recruited from healthy volunteers (Control group). The hypercholesterolemic patients were treated with a daily dose of 500 (n = 12) or 1000 mg (n = 27) of probu-
col (Probucol group). After overnight fasting, blood samples were drawn from a cubital vein once in the Control group and twice (at 0 and 4 weeks of treatment) in the Probucol group. We obtained informed consent from all participants at entry. This study was conducted in accordance with the Declaration of Helsinki [13]. 2.2. Lipids, apolipoproteins, lecithin-cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), angiopoietin-like protein 3 (ANGPTL3), and remnant-like particle cholesterol and triglyceride (RLP-C and RLP-TG) Fasting serum TC, TG, and PL concentrations were determined enzymatically using an automated analyzer. LDL-C and HDL-C concentrations were measured using homogenous methods with commercial kits (LDL-EX, Denka Seiken, Tokyo, Japan; Determiner L HDL, Kyowa Medex, Tokyo, Japan). Apolipoproteins were determined using a turbidimetric immunoassay (ApoA-I Auto N, ApoB Auto N, Daiichi Pure Chemicals, Tokyo, Japan). LCAT activity was expressed as the rate of reduction in the serum free cholesterol concentration during a 37 ◦ C-incubation with endogenous lipoproteins as substrate. CETP mass was measured using an enzyme immunoassay with a commercial kit (CETP ELISA Daiichi, Daiichi Pure Chemicals). ANGPTL3 was determined using a sandwich ELISA system in which mouse monoclonal antibody (OM-ANG-3C) was used as the first antibody and ANGPTL3 was detected using horseradish peroxidase (HRP)-conjugated rabbit polyclonal antibody (OP-ANG-2). These antibodies were raised against recombinant ANGPTL3 antigen (TSA 2306). Serum was diluted 11 times with dilution buffer for this assay and calculated by recombinant ANGPTL3. The detailed assay method, including normal control intervals, will be reported elsewhere. Remnant-like particles were separated using an immunoaffinity gel coupled with two monoclonal antibodies against apoAI and apoB100. The TC and TG concentrations in this fraction were defined as RLP-C and RLP-TG, respectively [14]. 2.3. Preβ1-HDL concentration and turnover rate Plasma was separated from EDTA-anticoagulated blood samples at 0 ◦ C using low-speed centrifugation because the pre1-HDL concentration changes significantly even at 4 ◦ C [15]. The plasma was then mixed with 20 volumes of 50% sucrose solution for stabilization. The pretreated samples were frozen at –80 ◦ C until the immunoassay was performed. The pre1-HDL concentration was determined using an enzyme-linked immunoassay with a specific monoclonal antibody (Daiichi Pure Chemicals). The turnover rate of the plasma pre1-HDL was quantified by measuring the interval required for half of the baseline pre1-HDL to mature into ␣-migrating HDL via LCAT
T. Miida et al. / Atherosclerosis 200 (2008) 329–335
activity (conversion half-time of pre1-HDL:CHTpre1 ) [16]. 2.4. HPLC The cholesterol and phospholipid profiles in serum lipoproteins were analyzed using a dual detection HPLC system with two tandem connected TSKgel LipopropakXL columns (300 mm × 7.8 mm; Tosoh, Tokyo, Japan) according to the method of Usui and coworkers [17] at Skylight Biotech (Akita, Japan). We quantified individual subfractions using the best curve fitting analysis, assuming that the particle sizes of all subfractions followed a Gaussian distribution. The particle sizes for individual subfractions were previously determined as 44.5–64 nm (large very low-density lipoprotein [VLDL]), 36.8 nm (medium VLDL), 31.3 nm (small VLDL), 28.6 nm (large low-density lipoprotein [LDL]), 25.5 nm (medium LDL), 23 nm (small LDL), 16.7–20.7 nm (very small LDL), 13.5–15 nm (very large high-density lipoprotein [HDL]), 12.1 nm (large HDL), 10.9 nm (medium HDL), 9.8 nm (small HDL), and 7.6–8.8 nm (very small HDL) [17]. 2.5. Inflammatory markers We used two inflammatory markers to evaluate mild chronic inflammation. The high-sensitivity C-reactive protein (hs-CRP) and serum amyloid A (SAA) were measured using latex agglutination assays with commercial kits (NANOPIA CRP, Daiichi Pure Chemicals; LZ Test SAA Eiken, Eiken Chemical, Tokyo, Japan). The detection limits of hs-CRP and SAA were 0.1 and 3 mg/L, respectively.
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2.6. Statistical methods The significance of the difference between the baseline and post-treatment values was assessed using the two-sided Student’s t-test or Wilcoxon signed-ranks test. The relationship between two variables was examined using Spearman’s rank correlation. For all analyses, p < 0.05 was considered statistically significant. Values are expressed as the mean ± S.D.
3. Results 3.1. Effects of probucol on lipids, apolipoproteins, and inflammatory markers The Probucol and Control groups were very similar in anthropometric variables and coronary risk factors other than the lipoprotein parameters (Table 1). The TC, PL, LDL-C, and apoB concentrations were greater in the Probucol group than in the Control group by 22, 15, 34, and 24%, respectively, whereas the HDL-C and apoAI concentrations did not differ between the two groups. After 4 weeks of probucol treatment, the percent reduction in the HDL-C concentration was three times greater than that in the LDL-C concentration (34% vs. 11%, Table 1). Corresponding to these changes, probucol also decreased TC, PL, apoB, and apoAI significantly, whereas it did not change the TG concentration. At baseline, the SAA and hs-CRP concentrations in the Probucol group were not elevated compared with those in the Control group. The 4-week probucol treatment reduced the SAA concentration significantly, but not hs-CRP (Table 1).
Table 1 Serum lipoprotein profile of the hypercholesterolemic patients before and after treatment with probucol Probucol (n = 39)
Age (years) M/F Body height (m) Body weight (kg) BMI (kg/m2 ) Hypertension (%) Current smoker (%) Diabetes (%) TC (mg/dL) TG (mg/dL) PL (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) ApoAI (mg/L) ApoB (mg/L) SAA (mg/L) CRP (mg/L)
Control (n = 39)
Baseline
Week 4
60.4 (9.7) 12/27 1.58 (0.09) 57.7 (9.2) 23.1 (2.3) 35.9 12.8 2.6 249 (23)*** 102 (35) 244 (21)*** 167 (26)*** 65 (17) 147 (22) 120 (14)*** 4.4 (2.6, 5.6) 0.54 (0.23, 0.84)
– – – – – – – – 207 (28)+++ 93 (54) 201 (25)*,+++ 149 (28)**,+++ 43 (12)***,+++ 110 (25)***,+++ 116 (17)***,+ 2.4 (1.9, 4.0)***,+++ 0.70 (0.36, 1.28)
60.2 (9.6) 12/27 1.57 (0.09) 57.1 (8.4) 22.9 (2.0) 20.5 17.9 0.0 204 (23) 91 (40) 213 (24) 125 (21) 61 (16) 141 (26) 97 (15) 4.9 (2.8, 7.3) 0.60 (0.40, 1.30)
BMI, body mass index; TC, total cholesterol; TG, triglyceride; PL, phospholipid; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; apoAI, apolipoprotein AI; apoB, apolipoprotein B; SAA, serum amyloid A protein; CRP, C-reactive protein. The data are presented as the mean (S.D.) or median (25th and 75th percentiles). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control group. + p < 0.05, +++ p < 0.001 vs. baseline.
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Table 2 Results of HPLC of the lipoprotein subfractions Probucol (n = 25) Baseline
Control (n = 6) Week 4
Cholesterol (mg/dL) VLDL (large) VLDL (medium) VLDL (small) LDL (large) LDL (medium) LDL (small) LDL (very small) HDL (very large) HDL (large) HDL (medium) HDL (small) HDL (very small)
15.8 18.7 15.1 40.0 51.1 29.2 15.2 4.9 14.9 20.3 16.0 9.5
± ± ± ± ± ± ± ± ± ± ± ±
6.5* 4.4*** 3.6* 6.6 8.0*** 6.8*** 3.9*** 3.5 11.7 4.5 2.5* 1.0***
10.9 15.0 12.1 31.8 47.3 31.0 16.5 2.4 5.0 14.8 14.0 7.9
± ± ± ± ± ± ± ± ± ± ± ±
6.8++ 4.6*,+++ 3.4+++ 7.4+++ 9.3**,+ 7.3*** 4.1***,+ 0.5*,++ 5.1*,+++ 5.9+++ 2.8+++ 1.4+++
9.9 10.5 11.8 35.0 40.4 20.4 10.0 5.4 17.3 18.4 13.1 7.3
± ± ± ± ± ± ± ± ± ± ± ±
4.5 3.5 3.1 6.3 2.5 2.0 1.3 2.1 7.8 6.4 2.8 0.9
Phospholipid (mg/dL) VLDL (large) VLDL (medium) VLDL (small) LDL (large) LDL (medium) LDL (small) LDL (very small) HDL (very large) HDL (large) HDL (medium) HDL (small) HDL (very small)
17.5 15.9 16.8 31.7 33.7 20.2 12.4 11.5 30.0 35.0 27.8 27.2
± ± ± ± ± ± ± ± ± ± ± ±
7.8 3.6*** 3.2* 4.2* 4.8 4.1 2.9* 9.9 14.7 5.4 3.3 2.7*
12.8 13.4 13.4 26.7 31.7 20.9 13.0 4.0 14.1 28.4 23.9 23.8
± ± ± ± ± ± ± ± ± ± ± ±
7.9+++ 4.2*,+++ 3.3+++ 5.0+++ 5.5+ 4.5 3.1 2.3**,+++ 10.3***,+++ 8.6+++ 4.5+++ 3.6+++
12.2 9.7 13.7 27.0 26.7 14.6 8.5 14.5 34.6 33.7 25.4 23.1
± ± ± ± ± ± ± ± ± ± ± ±
4.0 2.7 2.7 3.4 1.8 1.4 1.1 6.1 12.9 9.0 5.5 4.5
The lipoprotein subfractions were separated from fasting serum samples using a HPLC system. VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. The data are presented as the mean ± S.D. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control group. + p < 0.05, ++ p < 0.01, and +++ p < 0.001 vs. baseline.
The median value of the SAA at week 4 was even lower than that in the Control group. There was no significant correlation between the changes in SAA and reductions in HDL-C (r = 0.129). 3.2. Effects of probucol on the lipoprotein subfractions High-performance liquid chromatography revealed that probucol reduced VLDL, LDL, and HDL particles. In all
patients, the peak fraction of HDL particles shifted to a smaller subfraction at week 4 compared to baseline (data not shown). The percent reduction in PL was much greater for the very large and large HDL particles (65 and 51%) than for the smaller HDL particles (Table 2). For the very large HDL (the largest HDL subfraction), the percent reduction in PL was greater than that in cholesterol. In all of the VLDL subfractions, probucol reduced the cholesterol and PL concentrations slightly (16–30%), but
Table 3 Effect of probucol on RLP and pre1-HDL Probucol Baseline RLP-C (mg/dL) RLP-TG (mg/dL) RLP-C/RLP-TG Pre1-HDL (mg/L apoAI) Pre1-HDL (%) CHTpre1 (min)
(1.4)***
5.3 9.4 (8.2) 0.89 (0.55)** 21.7 (7.3) 1.5 (0.6) 41.5 (9.8)
Control Week 4 4.4 (2.2)*,+ 8.0 (6.7) 0.74 (0.36)* 24.7 (5.2)**,++ 2.4 (0.8)***,+++ 44.8 (9.9)
RLP-C, remnant-like particle cholesterol; RLP-TG, remnant-like particle triglyceride; CHTpre1 , conversion half time of pre1-HDL. The data are presented as the mean (S.D.). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control group. + p < 0.05, ++ p < 0.01, and +++ p < 0.001 vs. baseline.
3.3 (0.8) 9.1 (6.1) 0.45 (0.26) 19.5 (5.9) 1.4 (0.4) 45.3 (11.7)
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Table 4 Effect of probucol on HDL regulators in hypercholesterolemic patients Probucol Baseline LCAT (FC mmol/L/h) CETP mass (mg/L) AGPTL3 (g/L)
(26.7)***
125.7 2.21 (0.55) 145 (119, 154)
Control Week 4 107.7 (24.3)***,+++ 2.78 (0.70)***,+++ 113 (103, 134)+
90.2 (18.3) 1.90 (0.43) 119 (107, 144)
LCAT, lecithin-cholesterol acyltransferase; CETP cholesteryl ester transfer protein; AGPTL3, angiopoietin-like protein 3. The data are presented as the mean (S.D.) or median (25th, 75th percentiles). ** p < 0.01, *** p < 0.001 vs. Control group. + p < 0.05, +++ p < 0.001 vs. baseline.
significantly. Conversely, probucol reduced the large- and medium-sized LDL, but increased the small LDL particles slightly. 3.3. Effects of probucol on RLP-C and preβ1-HDL The RLP-C concentration was significantly higher at baseline in the Probucol group than in the Control group. Probucol slightly, but significantly, decreased the RLP-C concentration (Table 3). The RLP-C/RLP-TG ratio was greater in the Probucol group at baseline than in the Control group and did not change significantly after probucol treatment. Probucol increased lipid-poor HDL particles despite the marked reduction in HDL-C and PL. The absolute and relative concentrations of pre1-HDL increased by 13.8 and 60%, respectively (Table 3). CHTpre1 , a tentative marker of HDL maturation, did not get longer after probucol treatment, and the value at week 4 was very similar to that in the Control group. 3.4. Effects of probucol on HDL regulators Probucol changed the levels of key regulators of HDL metabolism (Table 4). At baseline, the LCAT activity was significantly higher in the Probucol group than in the Control group. At week 4, it decreased by 14%, but was still higher than in the Control group. The CETP mass increased by 26% after probucol treatment and was 1.5 times greater than in the Control group. Moreover, the median value of AGPTL3 decreased by 22%.
reduction in the HDL-PL concentration was greater for larger HDL particles than for smaller HDL particles (Table 2). Despite the 25% reduction in the serum apoAI, the pre1HDL concentration, expressed as the apoAI mass, increased significantly (Table 3), with a shift in the peak HDL diameter to smaller particles. Probucol is a well-known cholesterol-lowering agent that decreases HDL particles [6–8]. Similar to our results, Inazu et al. [7] reported that long-term probucol treatment markedly reduced HDL-C and HDL-PL, with a 23% increase in CETP mass. These reductions in HDL lipids were much greater for HDL2 (larger HDL particles) than for HDL3 (smaller particles). Our results (Table 4) are consistent with previous results in which immunoblotting for apoAI revealed that 4 weeks of probucol treatment resulted in a greater reduction in HDL2b (the largest HDL particle) than in HDL2a and HDL3 and that the CETP mass increased by 20% [6]. However, the actual transfer of cholesteryl ester (CE) mass from HDL to VLDL–LDL particles are dependent on these acceptor lipoprotein concentrations. In fact, probucol decreased the accentuated CE transfer to the normal level in one study [18], whereas it increased CE transfer in other studies [8,19]. Ikewaki et al. [20] measured the CETP mass in 591 Japanese subjects and examined the relationship between CETP mass and HDL-C; the correlation between them was significant, but very weak (r = − 0.173). We failed to detect a significant Table 5 Correlations of the changes in ANGPTL3 and CETP mass with those in HPLC-fractionated cholesterol and phospholipids ANGPTL3
3.5. Relationships between HDL regulators and HPLC-fractionated HDL lipids The change in ANGPTL3 concentration caused by probucol treatment had a significantly positive correlation with that in large HDL-PL. However, the change in CETP mass tended to be positively correlated with very small HDL-C (Table 5).
4. Discussion Probucol treatment decreased ANGPTL3 and HDL-PL while increasing pre1-HDL in patients with hypercholesterolemia. ANGPTL3 was decreased by 22% after a 4-week treatment with probucol (Table 4). HPLC indicated that the
CETP mass
Correlation coefficient
p
Correlation coefficient
p
Cholesterol HDL (very large) HDL (large) HDL (medium) HDL (small) HDL (very small)
0.248 0.240 0.198 0.084 0.302
N.S. N.S. N.S. N.S. N.S.
0.261 0.146 −0.128 0.239 0.375
N.S. N.S. N.S. N.S. 0.1
Phospholipids HDL (very large) HDL (large) HDL (medium) HDL (small) HDL (very small)
0.095 0.455 0.162 0.103 0.153
N.S. <0.05 N.S. N.S. N.S.
0.164 0.064 −0.127 0.067 0.335
N.S. N.S. N.S. N.S. N.S.
AGPTL3, angiopoietin-like protein 3; CETP, cholesteryl ester transfer protein.
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correlation between CETP mass and the HDL subfraction changes induced by probucol (Table 5). Therefore, it is not clear why probucol decreases both HDL-C and HDL-PL. In general, an extremely low HDL-C concentration is caused by either impaired maturation of HDL or enhanced hydrolysis of mature HDL. For example, patients with Tangier disease who completely lack ATP-binding cassette transporter A1 (ABCA1) activity and patients with LCAT gene mutations (LCAT deficiency or fish-eye disease) have HDL-C levels < 10 mg/dL [21-23]. In these patients, the relative pre1-HDL concentrations are greatly elevated and their pre1-HDL could not be converted into large spherical HDL (complete lack of pre1-HDL maturation). In hemodialysis patients, the LCAT activity was reduced to 65% of the normal level, whereas the pre1-HDL concentration increased by 126% and its maturation was severely impaired [24]. Because the probucol-treated patients had a normal CHTpre1 (a tentative marker of HDL maturation) despite the high pre1-HDL concentration (Table 3), the marked reduction in HDL-C is not likely attributable to impaired HDL maturation. Our results strongly suggest that ANGPTL3-mediated activation of EL promotes the hydrolysis of HDL-PL, resulting in a reduction in HDL-C and the generation of pre1-HDL in probucol-treated patients. Koishi et al. [25] first identified ANGPTL3 in hypolipidemic mutant mice (KK/San) using positional cloning; when ANGPTL3 was overexpressed or injected intravenously, the serum TC and TG increased significantly. Further research revealed that ANGPTL3 suppresses EL, which hydrolyzes HDL-PL via its phospholipase activity [11]. In a mouse model expressing human EL, there was an inverse relationship between the HDL-C level and EL expression [26]. Therefore, lowering ANGPTL3 is likely to enhance EL activity and subsequent HDL-PL hydrolysis and HDL reduction. In humans, the plasma ANGPTL3 mass had a significant positive correlation with the serum HDL-C and HDL-PL concentrations [11]. In probucol-treated patients, the change in ANGPTL3 was significantly correlated only with that in the large HDLPL (Table 5). If HDL hydrolysis is enhanced by EL in probucol-treated patients, more pre1-HDL can dissociate from large HDL particles as a surface remnant. Similar pre1-HDL generation is reported when plasma or HDL is hydrolyzed by hepatic lipase [4] or bacterial TG lipase [5]. These observations are comprehensible because the main pre1-HDL components are phospholipids and apoAI [2]. It is likely that ANGPTL3 suppression also induced the VLDL and RLP-C reductions in the probucol-treated patients. Probucol decreased the VLDL and RLP-C concentrations to a lesser extent than HDL (Table 2). ANGPTL3 inhibits not only EL, but also LPL. Furthermore, EL has relatively low TG lipase activity [27]. Therefore, it is likely that LPL is involved in the reduction of TG-rich lipoproteins in the probucol-treated patients. It is of great interest that RLP-C decreased significantly after probucol treatment
despite increasing CETP mass. In vitro, CETP enhances RLPC formation, whereas JTT705, a CETP inhibitor, prevents CE transfer to RLP [28]. Furthermore, HPLC revealed that RLP formation decreased after a fat load in patients with CETP deficiency [29]. These data suggest that CETP enhances RLPC formation by transferring CE from HDL. Although we did not measure LPL activity, the hydrolysis of RLP may overcome the CETP-mediated RLP formation in probucol-treated patients. Another interesting finding is that probucol decreased SAA, but not the hs-CRP concentration. We speculate that this anti-inflammatory effect of probucol is also related to the ANGPTL3-mediated activation of EL. SAA, an acute phase protein, is synthesized mainly in the liver. In addition to hepatocytes, endothelial cells, smooth muscle cells, and macrophages can secrete SAA. In human atherosclerotic lesions of the carotid and coronary arteries, SAA mRNA is expressed in most endothelial cells, some smooth muscle cells, and some macrophage-derived foam cells [30]. In cultured endothelial cells, tumor necrosis factor ␣ (TNF␣) induces VCAM1 expression. In human endothelial cells expressing EL, the hydrolysis of HDL by EL activates peroxisome proliferator-activated receptor ␣ (PPAR␣) and downstream target genes [31]. If this is the case in hypercholesterolemic patients, the probucol-induced suppression of ANGPTL3 causes EL activation in peripheral endothelial cells, resulting in the inhibition of local SAA synthesis by PPAR␣ activation. In summary, probucol decreases ANGPTL3 and HDL-PL while increasing pre1-HDL in patients with hypercholesterolemia. The overall effects of probucol on the development or regression of atherosclerosis remain to be determined. Acknowledgements This study was partly supported by the Grants-in-Aid of Science Research from the Ministry of Education, Science, and Culture of Japan (No. 16590815, 2004–2005). We are deeply indebted to Akemi Aoumi, Maiko Takeda, and Ayumi Hashimoto for their excellent technical assistance. References [1] Singh IM, Shishehbor MH, Ansell BJ. High-density lipoprotein as a therapeutic target: a systematic review. JAMA 2007;298:786–98. [2] Castro GR, Fielding CJ. Early incorporation of cell-derived cholesterol into pre--migrating high-density lipoprotein. Biochemistry 1988;27:25–9. [3] Kawano M, Miida T, Fielding CJ, Fielding PE. Quantitation of preHDL-dependent and nonspecific components of the total efflux of cellular cholesterol and phospholipid. Biochemistry 1993;32:5025–8. [4] Barrans A, Collet X, Barbaras R, et al. Hepatic lipase induces the formation of pre-1 high density lipoprotein (HDL) from triacylglycerol-rich HDL2. A study comparing liver perfusion to in vitro incubation with lipases. J Biol Chem 1994;269:11572–7. [5] Miida T, Sakai K, Ozaki K, et al. Bezafibrate increases pre1-HDL at the expense of HDL2b in hypertriglyceridemia. Arterioscler Thromb Vasc Biol 2000;20:2428–33.
T. Miida et al. / Atherosclerosis 200 (2008) 329–335 [6] Miida T, Yamaguchi T, Tsuda T, Okada M. High pre1-HDL levels in hypercholesterolemia are maintained by probucol but reduced by a low-cholesterol diet. Atherosclerosis 1998;138:129–34. [7] Inazu A, Koizumi J, Kajinami K, et al. Opposite effects on serum cholesteryl ester transfer protein levels between long-term treatments with pravastatin and probucol in patients with primary hypercholesterolemia and xanthoma. Atherosclerosis 1999;145:405–13. [8] Franceschini G, Sirtori M, Vaccarino V, et al. Mechanisms of HDL reduction after probucol. Changes in HDL subfractions and increased reverse cholesteryl ester transfer. Arteriosclerosis 1989;9:462–9. [9] Noto H, Kawamura M, Hashimoto Y, et al. Modulation of HDL metabolism by probucol in complete cholesteryl ester transfer protein deficiency. Atherosclerosis 2003;171:131–6. [10] Shimizugawa T, Ono M, Shimamura M, et al. ANGPTL3 decreases very low-density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase. J Biol Chem 2002;277:33742–8. [11] Shimamura M, Matsuda M, Yasumo H, et al. Angiopoietin-like protein3 regulates plasma HDL cholesterol through suppression of endothelial lipase. Arterioscler Thromb Vasc Biol 2007;27:366–72. [12] Hata Y, Mabuchi H, Saito Y, et al. Working Committee on JAS Guideline for Diagnosis and Treatment of Hyperlipidemias. Report of the Japan Atherosclerosis Society (JAS) Guideline for Diagnosis and Treatment of Hyperlipidemia in Japanese adults. J Atheroscler Thromb 2002;9:1–27. [13] Carlson RV, Boyd KM, Webb DJ. The revision of the Declaration of Helsinki: past, present and future. Br J Clin Pharmacol 2004;57:695–713. [14] Nakajima K, Saito T, Tamura A, et al. Cholesterol in remnant-like lipoproteins in human serum using monoclonal anti apo B-100 and anti apo A-I immunoaffinity mixed gel. Clin Chim Acta 1993;223:53–71. [15] Miida T, Miyazaki O, Nakamura Y, et al. Analytical performance of a sandwich enzyme immunoassay for pre1-HDL in stabilized plasma. J Lipid Res 2003;44:645–50. [16] Miida T, Obayashi K, Seino U, et al. LCAT-dependent conversion rate is a determinant of plasma pre1-HDL concentration in healthy Japanese. Clin Chim Acta 2004;350:107–14. [17] Okazaki M, Usui S, Ishigami M, et al. Identification of unique lipoprotein subclasses for visceral obesity by component analysis of cholesterol profile in high performance liquid chromatography. Arterioscler Thromb Vasc Biol 2005;25:578–84. [18] Bagdade JD, Kaufman D, Ritter MC, Subbaiah PV. Probucol treatment in hypercholesterolemic patients: effects on lipoprotein composition, HDL particle size, and cholesteryl ester transfer. Atherosclerosis 1990;84:145–54.
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[19] Matsuzawa Y, Yamashita S, Funahashi T, Yamamoto A, Tarui S. Selective reduction of cholesterol in HDL2 fraction by probucol in familial hypercholesterolemia and hyper-HDL2 cholesterolemia with abnormal cholesteryl ester transfer. Am J Cardiol 1988;62:66B– 72B. [20] Ikewaki K, Mabuchi H, Teramoto T, et al. Japan CETP Study Group. Association of cholesteryl ester transfer protein activity and TaqIB polymorphism with lipoprotein variations in Japanese subjects. Metabolism 2003;52:1564–70. [21] Asztalos BF, Brousseau ME, McNamara JR, et al. Subpopulations of high density lipoproteins in homozygous and heterozygous Tangier disease. Atherosclerosis 2001;156:217–25. [22] Asztalos BF, Schaefer EJ, Horvath KV, et al. Role of LCAT in HDL remodeling: investigation of LCAT deficiency states. J Lipid Res 2007;48:592–9. [23] Miida T, Zhang B, Obayashi K, et al. T13M mutation of lecithincholesterol acyltransferase gene causes fish-eye disease. Clin Chim Acta 2004;343:201–8. [24] Miida T, Miyazaki O, Hanyu O, et al. LCAT-dependent conversion of pre1-HDL into ␣-migrating HDL is severely delayed in hemodialysis patients. J Am Soc Nephrol 2003;14:732–8. [25] Koishi R, Ando Y, Ono M, et al. Angptl3 regulates lipid metabolism in mice. Nat Genet 2002;30:151–7. [26] Ishida T, Choi S, Kundu RK, et al. Endothelial lipase is a major determinant of HDL level. J Clin Invest 2003;111:347–55. [27] McCoy MG, Sun GS, Marchadier D, et al. Characterization of the lipolytic activity of endothelial lipase. J Lipid Res 2002;43: 921–9. [28] Okamoto H, Miyai A, Sasase T, et al. Cholesteryl ester transfer protein promotes the formation of cholesterol-rich remnant like lipoprotein particles in human plasma. Clin Chim Acta 2007;375:92–8. [29] Inazu A, Nakajima K, Nakano T, et al. Decreased post-prandial triglyceride response and diminished remnant lipoprotein formation in cholesteryl ester transfer protein (CETP) deficiency. Atherosclerosis, in press. [30] Meek RL, Urieli-Shoval S, Benditt EP. Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function. Proc Natl Acad Sci 1993;91:3186–90. [31] Ahmed W, Orasanu G, Nehra V, et al. High-density lipoprotein hydrolysis by endothelial lipase activates PPAR␣: a candidate mechanism for high-density lipoprotein-mediated repression of leukocyte adhesion. Circ Res 2006;98:490–8.
Probucol markedly reduces HDL phospholipids and elevated pre1-HDL without delayed conversion into ␣-migrating HDL: Putative role of angiopoietin-like protein 3 in probucol-induced HDL remodeling Takashi Miida a,∗ , Utako Seino b , Osamu Miyazaki c , Osamu Hanyu d , Satoshi Hirayama d , Toshikazu Saito e , Yuichi Ishikawa e , Suguru Akamatsu e , Toshimitsu Nakano e , Katsuyuki Nakajima e , Mitsuyo Okazaki f , Masahiko Okada b a
e
Department of Clinical Laboratory Medicine, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan b Division of Clinical Preventive Medicine, Department of Community Preventive Medicine, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi 1-757, Chuo-ku, Niigata, Niigata 951-8510, Japan c Diagnostics Research Laboratories, Daiichi Pure Chemicals, Koyodai 3-3-1, Ryugasaki, Ibaraki 301-0852, Japan d Division of Endocrinology and Metabolism, Department of Homeostatic Regulation and Developments, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi 1-757, Chuo-ku, Niigata, Niigata 951-8510, Japan Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Shinagawa Grand Central Tower, Konan 2-16-4, Minato-Ku, Tokyo 108-8242, Japan f Laboratory of Chemistry, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Kohnodai 2-8-30, Ichikawa, Chiba 272-0827, Japan Received 16 November 2007; accepted 27 December 2007 Available online 14 February 2008
Abstract Probucol is a unique hypolipidemic agent that increases cholesteryl ester transfer protein (CETP) activity. Enhanced CETP-mediated conversion of high-density lipoprotein (HDL) partly explains the probucol-induced decrease in HDL cholesterol and increase in plasma pre1-HDL (native lipid-poor HDL) concentrations. However, HDL cholesterol is reduced in patients that are completely deficient in CETP. Angiopoietin-like protein 3 (ANGPTL3) is an endogenous suppressor of endothelial lipase that promotes the hydrolysis of HDL phospholipids and may generate pre1-HDL. To determine whether probucol decreases ANGPTL3 and HDL phospholipids while increasing pre1-HDL, we measured these parameters before and after a 4-week probucol treatment in 39 hypercholesterolemic patients and age- and sex-matched controls. The median ANGPTL3 had decreased from 143 to 113 g/L by week 4 (p < 0.05). High-performance liquid chromatography revealed that probucol decreased the phospholipid content of very large (13.5–15 nm) and large (12.1 nm) HDL particles predominantly by 65% (p < 0.01) and 53% (p < 0.001), respectively. The change in ANGPTL3, but not CETP mass, was positively correlated with that in large HDL phospholipids (r = 0.455, p < 0.05). The absolute and relative concentrations of pre1-HDL increased by 14% (p < 0.01) and 60% (p < 0.001), respectively. The conversion rate of preβ1-HDL into α-migrating HDL by lecithin-cholesterol acyltransferase did not change significantly. In conclusion, probucol decreases plasma ANGPTL3 and HDL phospholipids while increasing pre1-HDL. We speculate that probucol induces HDL remodeling via an endothelial lipase-mediated pathway. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: ABCA1; Endothelial lipase; Inflammation; LCAT; Reverse cholesterol transport
1. Introduction
∗
Corresponding author. Tel.: +81-3-5802-1104; fax: +81-3-5684-1609. E-mail address: [email protected] (T. Miida).
0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.12.031
Numerous epidemiological studies clearly show that low high-density lipoprotein cholesterol (HDL-C) is a significant predictor of cardiovascular disease [1]. This favorable effect of HDL is related to its ability to remove excess
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cholesterol from peripheral tissues and transport it to the liver for further metabolism (reverse cholesterol transport; RCT). HDL consists of distinct particles that have various compositions and physiological functions; these can be classified into several HDL subfractions using different methods. Pre1-HDL is a distinct lipid-poor HDL Lp-AI subfraction (the lipoprotein contains apolipoprotein AI (apoAI), but no apoAII) [2]. Previously, we showed that pre1-HDL accelerates the cholesterol efflux from cultured fibroblasts in a dose-dependent manner [3]. Several lines of evidence suggest that plasma pre1-HDL is at least partly generated by the action of lipase [4,5]. Probucol is a mild cholesterol-lowering agent that is a strong antioxidant. Probucol treatment increases the pre1HDL concentration [6]. Because probucol increases both CETP mass and activity [6–8], the CETP-dependent conversion of large spherical HDL is most likely to contribute to the increase in pre1-HDL concentration in probucol-treated patients [6]. However, probucol reportedly even decreases HDL-C and HDL particle size in patients completely deficient in CETP [9], suggesting that it also affects HDL metabolism via a CETP-independent mechanism. Angiopoietin-like protein 3 (ANGPTL3) is a novel endogenous inhibitor of lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL), and endothelial lipase (EL) [10,11]. EL is a novel member of the lipase family that hydrolyzes HDL phospholipids (PL) [11]. During this process, pre1-HDL is likely generated from the surface remnants of hydrolyzed HDL. Therefore, probucol may promote HDL remodeling via the ANGPTL3-EL pathway. We examined whether probucol decreases ANGPTL3 and HDL-PL while increasing pre1-HDL in patients with hypercholesterolemia. To measure these parameters, we used our own enzyme-linked immunoassays for ANGPTL3 and pre1-HDL and a high-performance liquid chromatography (HPLC) system with analytical software for HDL phospholipids.
2. Materials and methods 2.1. Patients and study design We recruited study subjects from consecutive outpatients with hypercholesterolemia [total cholesterol (TC) concentration >5.69 mmol/L; 220 mg/dL] [12] at our institution. No patient had received a lipid-lowering agent previously. Routine laboratory tests revealed normal liver and kidney function. We excluded patients with a history of coronary heart disease or clinical symptoms of acute or chronic infection such as cough, sputum, and fever in the preceding 2 months. Thus, 39 patients aged 38–75 years were enrolled in the study. Age- and sex-matched control subjects were recruited from healthy volunteers (Control group). The hypercholesterolemic patients were treated with a daily dose of 500 (n = 12) or 1000 mg (n = 27) of probu-
col (Probucol group). After overnight fasting, blood samples were drawn from a cubital vein once in the Control group and twice (at 0 and 4 weeks of treatment) in the Probucol group. We obtained informed consent from all participants at entry. This study was conducted in accordance with the Declaration of Helsinki [13]. 2.2. Lipids, apolipoproteins, lecithin-cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), angiopoietin-like protein 3 (ANGPTL3), and remnant-like particle cholesterol and triglyceride (RLP-C and RLP-TG) Fasting serum TC, TG, and PL concentrations were determined enzymatically using an automated analyzer. LDL-C and HDL-C concentrations were measured using homogenous methods with commercial kits (LDL-EX, Denka Seiken, Tokyo, Japan; Determiner L HDL, Kyowa Medex, Tokyo, Japan). Apolipoproteins were determined using a turbidimetric immunoassay (ApoA-I Auto N, ApoB Auto N, Daiichi Pure Chemicals, Tokyo, Japan). LCAT activity was expressed as the rate of reduction in the serum free cholesterol concentration during a 37 ◦ C-incubation with endogenous lipoproteins as substrate. CETP mass was measured using an enzyme immunoassay with a commercial kit (CETP ELISA Daiichi, Daiichi Pure Chemicals). ANGPTL3 was determined using a sandwich ELISA system in which mouse monoclonal antibody (OM-ANG-3C) was used as the first antibody and ANGPTL3 was detected using horseradish peroxidase (HRP)-conjugated rabbit polyclonal antibody (OP-ANG-2). These antibodies were raised against recombinant ANGPTL3 antigen (TSA 2306). Serum was diluted 11 times with dilution buffer for this assay and calculated by recombinant ANGPTL3. The detailed assay method, including normal control intervals, will be reported elsewhere. Remnant-like particles were separated using an immunoaffinity gel coupled with two monoclonal antibodies against apoAI and apoB100. The TC and TG concentrations in this fraction were defined as RLP-C and RLP-TG, respectively [14]. 2.3. Preβ1-HDL concentration and turnover rate Plasma was separated from EDTA-anticoagulated blood samples at 0 ◦ C using low-speed centrifugation because the pre1-HDL concentration changes significantly even at 4 ◦ C [15]. The plasma was then mixed with 20 volumes of 50% sucrose solution for stabilization. The pretreated samples were frozen at –80 ◦ C until the immunoassay was performed. The pre1-HDL concentration was determined using an enzyme-linked immunoassay with a specific monoclonal antibody (Daiichi Pure Chemicals). The turnover rate of the plasma pre1-HDL was quantified by measuring the interval required for half of the baseline pre1-HDL to mature into ␣-migrating HDL via LCAT
T. Miida et al. / Atherosclerosis 200 (2008) 329–335
activity (conversion half-time of pre1-HDL:CHTpre1 ) [16]. 2.4. HPLC The cholesterol and phospholipid profiles in serum lipoproteins were analyzed using a dual detection HPLC system with two tandem connected TSKgel LipopropakXL columns (300 mm × 7.8 mm; Tosoh, Tokyo, Japan) according to the method of Usui and coworkers [17] at Skylight Biotech (Akita, Japan). We quantified individual subfractions using the best curve fitting analysis, assuming that the particle sizes of all subfractions followed a Gaussian distribution. The particle sizes for individual subfractions were previously determined as 44.5–64 nm (large very low-density lipoprotein [VLDL]), 36.8 nm (medium VLDL), 31.3 nm (small VLDL), 28.6 nm (large low-density lipoprotein [LDL]), 25.5 nm (medium LDL), 23 nm (small LDL), 16.7–20.7 nm (very small LDL), 13.5–15 nm (very large high-density lipoprotein [HDL]), 12.1 nm (large HDL), 10.9 nm (medium HDL), 9.8 nm (small HDL), and 7.6–8.8 nm (very small HDL) [17]. 2.5. Inflammatory markers We used two inflammatory markers to evaluate mild chronic inflammation. The high-sensitivity C-reactive protein (hs-CRP) and serum amyloid A (SAA) were measured using latex agglutination assays with commercial kits (NANOPIA CRP, Daiichi Pure Chemicals; LZ Test SAA Eiken, Eiken Chemical, Tokyo, Japan). The detection limits of hs-CRP and SAA were 0.1 and 3 mg/L, respectively.
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2.6. Statistical methods The significance of the difference between the baseline and post-treatment values was assessed using the two-sided Student’s t-test or Wilcoxon signed-ranks test. The relationship between two variables was examined using Spearman’s rank correlation. For all analyses, p < 0.05 was considered statistically significant. Values are expressed as the mean ± S.D.
3. Results 3.1. Effects of probucol on lipids, apolipoproteins, and inflammatory markers The Probucol and Control groups were very similar in anthropometric variables and coronary risk factors other than the lipoprotein parameters (Table 1). The TC, PL, LDL-C, and apoB concentrations were greater in the Probucol group than in the Control group by 22, 15, 34, and 24%, respectively, whereas the HDL-C and apoAI concentrations did not differ between the two groups. After 4 weeks of probucol treatment, the percent reduction in the HDL-C concentration was three times greater than that in the LDL-C concentration (34% vs. 11%, Table 1). Corresponding to these changes, probucol also decreased TC, PL, apoB, and apoAI significantly, whereas it did not change the TG concentration. At baseline, the SAA and hs-CRP concentrations in the Probucol group were not elevated compared with those in the Control group. The 4-week probucol treatment reduced the SAA concentration significantly, but not hs-CRP (Table 1).
Table 1 Serum lipoprotein profile of the hypercholesterolemic patients before and after treatment with probucol Probucol (n = 39)
Age (years) M/F Body height (m) Body weight (kg) BMI (kg/m2 ) Hypertension (%) Current smoker (%) Diabetes (%) TC (mg/dL) TG (mg/dL) PL (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) ApoAI (mg/L) ApoB (mg/L) SAA (mg/L) CRP (mg/L)
Control (n = 39)
Baseline
Week 4
60.4 (9.7) 12/27 1.58 (0.09) 57.7 (9.2) 23.1 (2.3) 35.9 12.8 2.6 249 (23)*** 102 (35) 244 (21)*** 167 (26)*** 65 (17) 147 (22) 120 (14)*** 4.4 (2.6, 5.6) 0.54 (0.23, 0.84)
– – – – – – – – 207 (28)+++ 93 (54) 201 (25)*,+++ 149 (28)**,+++ 43 (12)***,+++ 110 (25)***,+++ 116 (17)***,+ 2.4 (1.9, 4.0)***,+++ 0.70 (0.36, 1.28)
60.2 (9.6) 12/27 1.57 (0.09) 57.1 (8.4) 22.9 (2.0) 20.5 17.9 0.0 204 (23) 91 (40) 213 (24) 125 (21) 61 (16) 141 (26) 97 (15) 4.9 (2.8, 7.3) 0.60 (0.40, 1.30)
BMI, body mass index; TC, total cholesterol; TG, triglyceride; PL, phospholipid; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; apoAI, apolipoprotein AI; apoB, apolipoprotein B; SAA, serum amyloid A protein; CRP, C-reactive protein. The data are presented as the mean (S.D.) or median (25th and 75th percentiles). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control group. + p < 0.05, +++ p < 0.001 vs. baseline.
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Table 2 Results of HPLC of the lipoprotein subfractions Probucol (n = 25) Baseline
Control (n = 6) Week 4
Cholesterol (mg/dL) VLDL (large) VLDL (medium) VLDL (small) LDL (large) LDL (medium) LDL (small) LDL (very small) HDL (very large) HDL (large) HDL (medium) HDL (small) HDL (very small)
15.8 18.7 15.1 40.0 51.1 29.2 15.2 4.9 14.9 20.3 16.0 9.5
± ± ± ± ± ± ± ± ± ± ± ±
6.5* 4.4*** 3.6* 6.6 8.0*** 6.8*** 3.9*** 3.5 11.7 4.5 2.5* 1.0***
10.9 15.0 12.1 31.8 47.3 31.0 16.5 2.4 5.0 14.8 14.0 7.9
± ± ± ± ± ± ± ± ± ± ± ±
6.8++ 4.6*,+++ 3.4+++ 7.4+++ 9.3**,+ 7.3*** 4.1***,+ 0.5*,++ 5.1*,+++ 5.9+++ 2.8+++ 1.4+++
9.9 10.5 11.8 35.0 40.4 20.4 10.0 5.4 17.3 18.4 13.1 7.3
± ± ± ± ± ± ± ± ± ± ± ±
4.5 3.5 3.1 6.3 2.5 2.0 1.3 2.1 7.8 6.4 2.8 0.9
Phospholipid (mg/dL) VLDL (large) VLDL (medium) VLDL (small) LDL (large) LDL (medium) LDL (small) LDL (very small) HDL (very large) HDL (large) HDL (medium) HDL (small) HDL (very small)
17.5 15.9 16.8 31.7 33.7 20.2 12.4 11.5 30.0 35.0 27.8 27.2
± ± ± ± ± ± ± ± ± ± ± ±
7.8 3.6*** 3.2* 4.2* 4.8 4.1 2.9* 9.9 14.7 5.4 3.3 2.7*
12.8 13.4 13.4 26.7 31.7 20.9 13.0 4.0 14.1 28.4 23.9 23.8
± ± ± ± ± ± ± ± ± ± ± ±
7.9+++ 4.2*,+++ 3.3+++ 5.0+++ 5.5+ 4.5 3.1 2.3**,+++ 10.3***,+++ 8.6+++ 4.5+++ 3.6+++
12.2 9.7 13.7 27.0 26.7 14.6 8.5 14.5 34.6 33.7 25.4 23.1
± ± ± ± ± ± ± ± ± ± ± ±
4.0 2.7 2.7 3.4 1.8 1.4 1.1 6.1 12.9 9.0 5.5 4.5
The lipoprotein subfractions were separated from fasting serum samples using a HPLC system. VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. The data are presented as the mean ± S.D. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control group. + p < 0.05, ++ p < 0.01, and +++ p < 0.001 vs. baseline.
The median value of the SAA at week 4 was even lower than that in the Control group. There was no significant correlation between the changes in SAA and reductions in HDL-C (r = 0.129). 3.2. Effects of probucol on the lipoprotein subfractions High-performance liquid chromatography revealed that probucol reduced VLDL, LDL, and HDL particles. In all
patients, the peak fraction of HDL particles shifted to a smaller subfraction at week 4 compared to baseline (data not shown). The percent reduction in PL was much greater for the very large and large HDL particles (65 and 51%) than for the smaller HDL particles (Table 2). For the very large HDL (the largest HDL subfraction), the percent reduction in PL was greater than that in cholesterol. In all of the VLDL subfractions, probucol reduced the cholesterol and PL concentrations slightly (16–30%), but
Table 3 Effect of probucol on RLP and pre1-HDL Probucol Baseline RLP-C (mg/dL) RLP-TG (mg/dL) RLP-C/RLP-TG Pre1-HDL (mg/L apoAI) Pre1-HDL (%) CHTpre1 (min)
(1.4)***
5.3 9.4 (8.2) 0.89 (0.55)** 21.7 (7.3) 1.5 (0.6) 41.5 (9.8)
Control Week 4 4.4 (2.2)*,+ 8.0 (6.7) 0.74 (0.36)* 24.7 (5.2)**,++ 2.4 (0.8)***,+++ 44.8 (9.9)
RLP-C, remnant-like particle cholesterol; RLP-TG, remnant-like particle triglyceride; CHTpre1 , conversion half time of pre1-HDL. The data are presented as the mean (S.D.). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control group. + p < 0.05, ++ p < 0.01, and +++ p < 0.001 vs. baseline.
3.3 (0.8) 9.1 (6.1) 0.45 (0.26) 19.5 (5.9) 1.4 (0.4) 45.3 (11.7)
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Table 4 Effect of probucol on HDL regulators in hypercholesterolemic patients Probucol Baseline LCAT (FC mmol/L/h) CETP mass (mg/L) AGPTL3 (g/L)
(26.7)***
125.7 2.21 (0.55) 145 (119, 154)
Control Week 4 107.7 (24.3)***,+++ 2.78 (0.70)***,+++ 113 (103, 134)+
90.2 (18.3) 1.90 (0.43) 119 (107, 144)
LCAT, lecithin-cholesterol acyltransferase; CETP cholesteryl ester transfer protein; AGPTL3, angiopoietin-like protein 3. The data are presented as the mean (S.D.) or median (25th, 75th percentiles). ** p < 0.01, *** p < 0.001 vs. Control group. + p < 0.05, +++ p < 0.001 vs. baseline.
significantly. Conversely, probucol reduced the large- and medium-sized LDL, but increased the small LDL particles slightly. 3.3. Effects of probucol on RLP-C and preβ1-HDL The RLP-C concentration was significantly higher at baseline in the Probucol group than in the Control group. Probucol slightly, but significantly, decreased the RLP-C concentration (Table 3). The RLP-C/RLP-TG ratio was greater in the Probucol group at baseline than in the Control group and did not change significantly after probucol treatment. Probucol increased lipid-poor HDL particles despite the marked reduction in HDL-C and PL. The absolute and relative concentrations of pre1-HDL increased by 13.8 and 60%, respectively (Table 3). CHTpre1 , a tentative marker of HDL maturation, did not get longer after probucol treatment, and the value at week 4 was very similar to that in the Control group. 3.4. Effects of probucol on HDL regulators Probucol changed the levels of key regulators of HDL metabolism (Table 4). At baseline, the LCAT activity was significantly higher in the Probucol group than in the Control group. At week 4, it decreased by 14%, but was still higher than in the Control group. The CETP mass increased by 26% after probucol treatment and was 1.5 times greater than in the Control group. Moreover, the median value of AGPTL3 decreased by 22%.
reduction in the HDL-PL concentration was greater for larger HDL particles than for smaller HDL particles (Table 2). Despite the 25% reduction in the serum apoAI, the pre1HDL concentration, expressed as the apoAI mass, increased significantly (Table 3), with a shift in the peak HDL diameter to smaller particles. Probucol is a well-known cholesterol-lowering agent that decreases HDL particles [6–8]. Similar to our results, Inazu et al. [7] reported that long-term probucol treatment markedly reduced HDL-C and HDL-PL, with a 23% increase in CETP mass. These reductions in HDL lipids were much greater for HDL2 (larger HDL particles) than for HDL3 (smaller particles). Our results (Table 4) are consistent with previous results in which immunoblotting for apoAI revealed that 4 weeks of probucol treatment resulted in a greater reduction in HDL2b (the largest HDL particle) than in HDL2a and HDL3 and that the CETP mass increased by 20% [6]. However, the actual transfer of cholesteryl ester (CE) mass from HDL to VLDL–LDL particles are dependent on these acceptor lipoprotein concentrations. In fact, probucol decreased the accentuated CE transfer to the normal level in one study [18], whereas it increased CE transfer in other studies [8,19]. Ikewaki et al. [20] measured the CETP mass in 591 Japanese subjects and examined the relationship between CETP mass and HDL-C; the correlation between them was significant, but very weak (r = − 0.173). We failed to detect a significant Table 5 Correlations of the changes in ANGPTL3 and CETP mass with those in HPLC-fractionated cholesterol and phospholipids ANGPTL3
3.5. Relationships between HDL regulators and HPLC-fractionated HDL lipids The change in ANGPTL3 concentration caused by probucol treatment had a significantly positive correlation with that in large HDL-PL. However, the change in CETP mass tended to be positively correlated with very small HDL-C (Table 5).
4. Discussion Probucol treatment decreased ANGPTL3 and HDL-PL while increasing pre1-HDL in patients with hypercholesterolemia. ANGPTL3 was decreased by 22% after a 4-week treatment with probucol (Table 4). HPLC indicated that the
CETP mass
Correlation coefficient
p
Correlation coefficient
p
Cholesterol HDL (very large) HDL (large) HDL (medium) HDL (small) HDL (very small)
0.248 0.240 0.198 0.084 0.302
N.S. N.S. N.S. N.S. N.S.
0.261 0.146 −0.128 0.239 0.375
N.S. N.S. N.S. N.S. 0.1
Phospholipids HDL (very large) HDL (large) HDL (medium) HDL (small) HDL (very small)
0.095 0.455 0.162 0.103 0.153
N.S. <0.05 N.S. N.S. N.S.
0.164 0.064 −0.127 0.067 0.335
N.S. N.S. N.S. N.S. N.S.
AGPTL3, angiopoietin-like protein 3; CETP, cholesteryl ester transfer protein.
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correlation between CETP mass and the HDL subfraction changes induced by probucol (Table 5). Therefore, it is not clear why probucol decreases both HDL-C and HDL-PL. In general, an extremely low HDL-C concentration is caused by either impaired maturation of HDL or enhanced hydrolysis of mature HDL. For example, patients with Tangier disease who completely lack ATP-binding cassette transporter A1 (ABCA1) activity and patients with LCAT gene mutations (LCAT deficiency or fish-eye disease) have HDL-C levels < 10 mg/dL [21-23]. In these patients, the relative pre1-HDL concentrations are greatly elevated and their pre1-HDL could not be converted into large spherical HDL (complete lack of pre1-HDL maturation). In hemodialysis patients, the LCAT activity was reduced to 65% of the normal level, whereas the pre1-HDL concentration increased by 126% and its maturation was severely impaired [24]. Because the probucol-treated patients had a normal CHTpre1 (a tentative marker of HDL maturation) despite the high pre1-HDL concentration (Table 3), the marked reduction in HDL-C is not likely attributable to impaired HDL maturation. Our results strongly suggest that ANGPTL3-mediated activation of EL promotes the hydrolysis of HDL-PL, resulting in a reduction in HDL-C and the generation of pre1-HDL in probucol-treated patients. Koishi et al. [25] first identified ANGPTL3 in hypolipidemic mutant mice (KK/San) using positional cloning; when ANGPTL3 was overexpressed or injected intravenously, the serum TC and TG increased significantly. Further research revealed that ANGPTL3 suppresses EL, which hydrolyzes HDL-PL via its phospholipase activity [11]. In a mouse model expressing human EL, there was an inverse relationship between the HDL-C level and EL expression [26]. Therefore, lowering ANGPTL3 is likely to enhance EL activity and subsequent HDL-PL hydrolysis and HDL reduction. In humans, the plasma ANGPTL3 mass had a significant positive correlation with the serum HDL-C and HDL-PL concentrations [11]. In probucol-treated patients, the change in ANGPTL3 was significantly correlated only with that in the large HDLPL (Table 5). If HDL hydrolysis is enhanced by EL in probucol-treated patients, more pre1-HDL can dissociate from large HDL particles as a surface remnant. Similar pre1-HDL generation is reported when plasma or HDL is hydrolyzed by hepatic lipase [4] or bacterial TG lipase [5]. These observations are comprehensible because the main pre1-HDL components are phospholipids and apoAI [2]. It is likely that ANGPTL3 suppression also induced the VLDL and RLP-C reductions in the probucol-treated patients. Probucol decreased the VLDL and RLP-C concentrations to a lesser extent than HDL (Table 2). ANGPTL3 inhibits not only EL, but also LPL. Furthermore, EL has relatively low TG lipase activity [27]. Therefore, it is likely that LPL is involved in the reduction of TG-rich lipoproteins in the probucol-treated patients. It is of great interest that RLP-C decreased significantly after probucol treatment
despite increasing CETP mass. In vitro, CETP enhances RLPC formation, whereas JTT705, a CETP inhibitor, prevents CE transfer to RLP [28]. Furthermore, HPLC revealed that RLP formation decreased after a fat load in patients with CETP deficiency [29]. These data suggest that CETP enhances RLPC formation by transferring CE from HDL. Although we did not measure LPL activity, the hydrolysis of RLP may overcome the CETP-mediated RLP formation in probucol-treated patients. Another interesting finding is that probucol decreased SAA, but not the hs-CRP concentration. We speculate that this anti-inflammatory effect of probucol is also related to the ANGPTL3-mediated activation of EL. SAA, an acute phase protein, is synthesized mainly in the liver. In addition to hepatocytes, endothelial cells, smooth muscle cells, and macrophages can secrete SAA. In human atherosclerotic lesions of the carotid and coronary arteries, SAA mRNA is expressed in most endothelial cells, some smooth muscle cells, and some macrophage-derived foam cells [30]. In cultured endothelial cells, tumor necrosis factor ␣ (TNF␣) induces VCAM1 expression. In human endothelial cells expressing EL, the hydrolysis of HDL by EL activates peroxisome proliferator-activated receptor ␣ (PPAR␣) and downstream target genes [31]. If this is the case in hypercholesterolemic patients, the probucol-induced suppression of ANGPTL3 causes EL activation in peripheral endothelial cells, resulting in the inhibition of local SAA synthesis by PPAR␣ activation. In summary, probucol decreases ANGPTL3 and HDL-PL while increasing pre1-HDL in patients with hypercholesterolemia. The overall effects of probucol on the development or regression of atherosclerosis remain to be determined. Acknowledgements This study was partly supported by the Grants-in-Aid of Science Research from the Ministry of Education, Science, and Culture of Japan (No. 16590815, 2004–2005). We are deeply indebted to Akemi Aoumi, Maiko Takeda, and Ayumi Hashimoto for their excellent technical assistance. References [1] Singh IM, Shishehbor MH, Ansell BJ. High-density lipoprotein as a therapeutic target: a systematic review. JAMA 2007;298:786–98. [2] Castro GR, Fielding CJ. Early incorporation of cell-derived cholesterol into pre--migrating high-density lipoprotein. Biochemistry 1988;27:25–9. [3] Kawano M, Miida T, Fielding CJ, Fielding PE. Quantitation of preHDL-dependent and nonspecific components of the total efflux of cellular cholesterol and phospholipid. Biochemistry 1993;32:5025–8. [4] Barrans A, Collet X, Barbaras R, et al. Hepatic lipase induces the formation of pre-1 high density lipoprotein (HDL) from triacylglycerol-rich HDL2. A study comparing liver perfusion to in vitro incubation with lipases. J Biol Chem 1994;269:11572–7. [5] Miida T, Sakai K, Ozaki K, et al. Bezafibrate increases pre1-HDL at the expense of HDL2b in hypertriglyceridemia. Arterioscler Thromb Vasc Biol 2000;20:2428–33.
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