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Institut National de la Santé et de la Recherche Médicale Unité 551, Dyslipoproteinemia and Atherosclerosis (M.G., W.L.G., E.F., S.S., D.M., M.J.C.), and Service d’Endocrinologie-Métabolisme (E.B.), Hôpital de la Pitié, 75651 Paris, France
Address all correspondence and requests for reprints to: Dr. Maryse Guerin, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 551, Hôpital de la Pitié, Pavillon Benjamin Delessert, 83, Boulevard de l’Hôpital, 75651 Paris Cedex 13, France. E-mail: mguerin{at}infobiogen.fr.
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Abstract |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The past decade afforded new insight into the mechanism of action of fibrates on lipid and lipoprotein metabolism. Fibrates are agonists of peroxisome proliferator-activated receptor (PPAR)-
, specific transcription factors belonging to the nuclear hormone receptor superfamily. Activation of PPAR- by fibrates in tissues corresponding to major sites of fatty acid metabolism, such as the liver, adipose tissue, and macrophage, modulate the expression of several key genes encoding proteins involved in lipid metabolism. Indeed, fibrates induce up-regulation of genes encoding apoAI and apoAII in the liver (15, 16). Equally, activation of PPAR- by fibrates induces hepatic expression of LPL and thus enhances intravascular lipolysis of triglyceride-rich lipoprotein particles (17). More recently, it has been demonstrated that the expression of the CLA1 gene in human monocyte-macrophages can be induced by fibrate-activated PPAR- (18). Because the scavenger receptor class B, type I (SR-BI)/CLA-1 receptor is involved not only in HDL-mediated removal of cholesterol from peripheral cells but also in hepatic cholesterol uptake from HDL, fibrates may enhance both cellular cholesterol efflux and the reverse cholesterol transport pathway. In parallel, fibrates also induce 1) hepatic fatty acid uptake and ß- oxidation, resulting in reduction in triglyceride synthesis and production of VLDL by the liver (19); and 2) reduction in CE transfer from HDL to atherogenic VLDL, IDL, and LDL as a consequence of reduction in apoB-containing lipoprotein acceptors. This reduction thereby results in the normalization of intravascular CETP-mediated remodeling of triglyceride-rich lipoprotein particles and enhancing their removal from plasma (10, 12).
In the present study, we evaluated the effect of a potent fibrate, ciprofibrate, on the plasma profile of atherogenic apoB-containing lipoprotein subspecies and on apoAI-containing HDL subspecies; on CETP-mediated CE transfer from HDL to apoB-containing lipoproteins; and on plasma-mediated cellular cholesterol efflux in 10 patients presenting with the atherogenic IIB phenotype. The action of ciprofibrate resulted in 1) marked reduction of plasma VLDL-1, VLDL-2, and dense LDL levels, including a shift of the dense LDL subclass profile to more buoyant particles with attenuation of the atherogenic lipoprotein profile; 2) reduction of CETP-mediated CE transfer from HDL, primarily involving CE transfer from HDL to VLDL-1 and VLDL-2; and 3) stimulation of HDL-mediated cellular free cholesterol efflux through the cellular SR-BI receptor pathway.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Ten males (ages, 32–50 yr; mean, 40 ± 6 yr) who displayed the type IIB phenotype typical of combined or mixed hyperlipidemia, i.e. fasting plasma levels of cholesterol at least 6.0 mmol/liter; triglycerides at least 1.70 mmol/liter; and apoB at least 140 mg/dl, were selected for the study (20). All subjects exhibited levels of lipoprotein(a) less than 50 mg/dl (mean, 15 ± 5 mg/dl). Patients were excluded if they displayed dysbetalipoproteinemia, diabetes mellitus, secondary causes of hyperlipidemia such as hypothyroidism, renal impairment or nephrotic syndrome, or known liver or muscle disease. Other exclusion criteria included uncontrolled hypertension or any major cardiovascular event (myocardial infarction, severe or unstable angina pectoris, angioplasty, or cardiovascular surgery). None of the subjects was obese (body mass index < 30 kg/m2). Patients had ceased taking lipid-lowering drugs and had signed an informed consent form 6 wk before active treatment. The 6-wk period before treatment corresponded to a placebo period involving dietary stabilization (American Heart Association, step 1 diet). At wk 0, subjects started a 6-wk active period of treatment during which they received 100 mg/d ciprofibrate in capsule form before dinner. All patients tolerated the drug well, without any dropout. Blood samples (50 ml) were obtained by venipuncture of the brachial vein after an overnight fast at the time of inclusion into the study and after 6 wk of ciprofibrate (100 mg/d) treatment. Blood was collected in sterile EDTA-containing tubes (final concentration, 1 mg/ml), and plasma was immediately separated from blood cells by low-speed centrifugation at 2500 rpm for 20 min at 4 C.
The study was performed in accordance with the ethical principles set forth in the Declaration of Helsinki. The study protocol and amendment were reviewed and approved by an Ethics Committee and met national institutional requirements.
Lipid and protein analyses
The lipid content of plasma and isolated lipoprotein fractions were quantified enzymatically, using Boehringer Mannheim kits (Meylan, France), for total cholesterol (TC) and free cholesterol (FC). CE mass was calculated as (TC - FC) x 1.67 and thus represents the sum of the esterified cholesterol and fatty acid moieties (21). Bio-Mérieux kits (Marcy-l’Etoile, France) were used for determination of triglycerides and phospholipids. The bicinchoninic acid assay reagent (Pierce, Rockford, IL) was used for protein quantification. Lipoprotein mass was calculated as the sum of the mass of the individual lipid and protein components for each lipoprotein fraction. Plasma LDL-cholesterol was calculated using the Friedewald formula. Plasma apoAI and apoB concentrations were determined by immunodiffusion using commercial gels (Sebia, Issy-les-Moulineaux, France).
Isolation of plasma lipoprotein subfractions
Subfractions of triglyceride-rich lipoproteins, i.e. VLDL-1 (Sf 60–400), VLDL-2 (Sf 20–60), and IDL (Sf 12–20), were isolated from plasma (2 ml) by cumulative flotation after nonequilibrium density-gradient ultracentrifugation using a Beckman SW41 Ti rotor (Beckman Coulter, Fullerton, CA) (12). LDL and HDL subfractions were isolated from plasma (3 ml) by isopycnic density gradient ultracentrifugation at 40,000 rpm for 44 h at 15 C by minor modification of the method of Chapman et al. (21), as previously described (6). With this procedure, LDL were separated into five LDL subfractions (LDL-1, d 1.019–1.023 g/ml; LDL-2, d 1.023–1.029 g/ml; LDL-3, d 1.029–1.039 g/ml; LDL-4, d 1.039–1.050 g/ml; and LDL-5, d 1.050–1.063 g/ml) and five HDL subfractions (HDL-2b, d 1.063–1.091 g/ml; HDL-2a, d 1.091–1.110 g/ml; HDL-3a, d 1.110–1.133 g/ml; HDL-3b, d 1.133–1.156 g/ml; and HDL-3c, d 1.156–1.179 g/ml). After centrifugation, all lipoprotein subfractions were collected from the meniscus of the tube downward by aspiration with a precision micropipette (Gilson, Villier Le Bel, France) in aliquots of 0.8 ml, with the exception of the HDL-2b subfraction that was collected in an aliquot of 1.2 ml.
Determination of CE transfer from HDL to apoB-containing lipoproteins
Determination of CE transfer from HDL to apoB-containing lipoproteins was assayed by modification of the method of Guérin et al. (22), which exclusively involves physiological CE transfer between endogenous lipoprotein donor and acceptor particles in plasma from each patient (22). Radiolabeled HDL were obtained from the d > 1.063 g/ml fraction of each patient’s plasma, as previously described (22). CE transfer was determined after incubation of whole plasma from individual subjects at 37 C or 0 C for 3 h in the presence of radiolabeled HDL (equivalent to 1% of the total HDL-CE mass present in 1 ml of each subject’s plasma) and iodoacetamide (final concentration, 1.5 mmol/liter). After incubation, triglyceride-rich lipoproteins, i.e. VLDL-1, VLDL-2, and IDL, were isolated by ultracentrifugation, as described above. The total LDL (d 1.019–1.063 g/ml) and HDL (d > 1.063 g/ml) fractions were subsequently isolated by ultracentrifugation at 45,000 rpm for 24 h. The radioactive CE content of each isolated lipoprotein fraction was quantified by liquid scintillation counting. The rate of CE transfer was calculated from the known specific radioactivity of radiolabeled HDL-CE after its addition to plasma and is expressed in micrograms of CE transferred per hour per milliliter plasma (22).
Free cholesterol efflux from Fu5AH rat hepatoma cells
The capacity of each plasma sample to facilitate cellular cholesterol efflux from rat Fu5AH hepatoma cells was assayed according to the method of de la Llera Moya et al. (23). This experimental system has been validated as a model for efflux from cells of peripheral tissues via the SR-BI receptor (24). Briefly, cells were maintained in Eagle’s MEM containing 5% newborn calf serum and plated on 2.4-cm multiwell plates using 2 ml/well, i.e. 40,000 Fu5AH cells per well. Two days after plating, cellular cholesterol was labeled by incubation for 48 h with [3H]cholesterol (1 µCi/well). To allow equilibration of the label, the cells were washed twice with PBS and incubated for 24 h in MEM containing 0.5% BSA. Plated cells were subsequently incubated at 37 C, with 5% plasma diluted with MEM or with a 5% dilution of a d > 1.063 g/ml fraction depleted in apoB-containing lipoproteins (isolated from plasma after centrifugation at 40,000 rpm for 24 h at 15 C), or with a 5% dilution of a d > 1.125 g/ml fraction depleted in HDL-2 particles (isolated from the d > 1.063 g/ml fraction by ultracentrifugation at 40,000 rpm for 48 h at 15 C). After 4 h of incubation, the medium was collected, and the labeled cell cholesterol released was measured in an aliquot of the medium by liquid scintillation counting (Rackbeta 1209, Wallac, Turku, Finland). Triplicate wells of cells were also incubated with cholesterol acceptor-free medium to measure background (nonacceptor-mediated) cholesterol efflux. Before the start of the cholesterol efflux assay, a multiwell plate of cells was washed two times with PBS; cellular lipids were extracted by isopropanol; and cell radioactivity at time zero was measured by liquid scintillation counting. Cholesterol efflux (expressed as a percentage) was calculated as the amount of the label recovered in the medium divided by the total label in the cells at time zero. The background cholesterol efflux obtained in the absence of any acceptor (<2%) was subtracted from the efflux values obtained with the test samples.
Free cholesterol efflux from J774 mouse macrophage cells
The capacity of each plasma sample to facilitate cellular cholesterol efflux was equally determined in vitro with mouse J774 macrophage-like cells, which express ATP-binding cassette transporter-1 (ABCA1) at high levels, by the method of Sakr et al. (25). The J774 mouse macrophage cell system expresses SR-BI at very low levels (26). Briefly, cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and containing 1% L-glutamine and 0.75% penicillin-streptomycin. For efflux experiments, cells in DMEM containing 10% FBS were plated on 2.4-cm multiwell plates using 2 ml/well (100,000 cells per well). Three days after plating, cellular cholesterol was labeled by incubation of the cells for 24 h with [3H]cholesterol (1 µCi/well) in the culture media containing 2.5% FBS. To allow equilibration of the label among the various pools of cell cholesterol, cells were washed two times with PBS and incubated for 24 h in FBS-free culture medium containing 0.2% BSA. After this equilibration period, cells were washed with PBS and then incubated for 24 h in FBS-free culture medium with or without 0.2 mmol/liter 8-(4-chlorophenylthio)-cAMP. 8-(4-Chlorophenylthio)-cAMP was dissolved in distilled water at 50 mmol/liter and stored at -20 C. At the end of the pretreatment period with cAMP, cells were washed with PBS and incubated for 24 h with 1 ml of culture medium containing 2.5% or 5% plasma samples. After incubation, the medium was centrifuged at 2500 rpm for 10 min to remove floating cells and counted by liquid scintillation counting. Triplicate wells of cells were also incubated with cholesterol acceptor-free medium to measure background (nonacceptor-mediated) cholesterol efflux. Before the start of the cholesterol efflux assay, a multiwell plate of control or treated cells was washed with PBS, and cell radioactivity at time zero was measured by liquid scintillation counting. Cholesterol efflux was calculated as the amount of the label recovered in the medium divided by the total label in the cells at time zero and expressed as a percentage. The background cholesterol efflux obtained in the absence of any acceptor (<3%) was subtracted from the efflux values obtained with the test samples.
Statistical analysis
All data are presented as means ± SEM. The effects of ciprofibrate on plasma lipid levels, the plasma concentrations of lipoprotein subfractions, CE transfer from HDL to apoB-containing lipoproteins, and cellular free cholesterol efflux from cells were determined by comparing these parameters at the time of inclusion into the study with those after 6 wk of drug therapy by ANOVA, using Student’s paired t test. The level of statistical significance was taken as P < 0.05.
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Results |
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Plasma lipid and apolipoprotein levels before and 6 wk after ciprofibrate treatment at doses of 100 mg/d in type IIB patients (n = 10) are presented in Table 1. At baseline, all subjects displayed plasma lipid and apoB levels characteristic of the IIB phenotype, as defined in Patients and Methods. After fibrate therapy, we observed significant reductions in both plasma total cholesterol (-20%; P = 0.002) and triglyceride (-45%; P = 0.003) levels. In addition, drug therapy significantly lowered plasma VLDL-cholesterol (-42%; P = 0.007), LDL-cholesterol (-20%; P = 0.002), and apoB (-26%; P = 0.001) concentrations. Moreover, no significant changes in HDL-cholesterol or apoAI concentrations were detected in ciprofibrate-treated patients, although there was an overall trend toward increase in both HDL-cholesterol and apoAI levels (Table 1). Nonetheless, total HDL mass was increased (see below). Interestingly, patients presenting the IIB phenotype displayed subnormal levels of HDL-cholesterol (0.96 ± 0.08 mmol/liter) compared with normolipidemic subjects (1.37 ± 0.05 mmol/liter) of similar age and sex (27).
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A marked mean reduction (-25%; P = 0.0006) in total plasma triglyceride-rich apoB-containing lipoprotein concentrations (VLDL-1 + VLDL-2 + IDL) was observed in type IIB patients (324 ± 17 and 243 ± 10 mg/dl before and after ciprofibrate treatment, respectively). Plasma VLDL-1 concentrations were significantly lowered by 40% (P = 0.001) after ciprofibrate therapy (145 ± 13 and 87 ± 11 mg/dl before and after ciprofibrate, respectively). In addition, a reduction of 25% (P = 0.003) was seen in plasma VLDL-2 (112 ± 11 and 85 ± 7 mg/dl before and after ciprofibrate, respectively). By contrast, IDL levels were not significantly influenced by fibrate therapy (67 ± 6 and 72 ± 4 mg/dl before and after treatment, respectively).
On fibrate treatment, mean plasma LDL concentration was reduced by 17% (P = 0.005) (502 ± 16 and 418 ± 24 mg/dl before and after treatment, respectively). The density distribution of the mass of LDL subspecies (Fig. 2) revealed that dense LDL subfractions (LDL-4 and LDL-5) predominated in IIB patients before treatment, accounting for 51% of total LDL mass, whereas larger, lighter LDL particles of intermediate density (LDL-3) represented a minor component (27%) of the LDL mass profile. After fibrate treatment, the LDL profile was normalized, and peak density shifted toward the more buoyant, CE-rich LDL-3 subfraction. Indeed, we observed a significant increase in both the mean plasma concentration (+27%; P = 0.04) and the relative proportion (+41%; P < 0.0001) of LDL-3; concomitantly, significant reductions in plasma levels of both LDL-4 (-46%; P < 0.0001) and LDL-5 (-46%; P < 0.0001) and in the relative proportion of dense LDL subfractions (LDL-4 + LDL-5, -34%; P < 0.0001) were found.
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. Interestingly, no significant variations in plasma HDL-2 levels were observed in subjects taking drug therapy (125 ± 8 and 127 ± 12 mg/dl before and after fibrate, respectively), whereas fibrate treatment induced significant elevation (+22%; P = 0.009) in HDL-3 levels. Moreover, when individual HDL particle subfractions were considered, we detected a significant increase in HDL-3a (+13%; P < 0.02), HDL-3b (+22%; P < 0.02), and HDL-3c (+48%; P < 0.02) concentrations.
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Table 2 shows the effect of ciprofibrate on the transfer rate of CEs from HDL to individual apoB-containing lipoproteins in the plasma of type IIB patients. After fibrate treatment with ciprofibrate at 100 mg/d, the mean rate of transfer of CEs from HDL to apoB-containing lipoproteins was significantly reduced (-25%; P = 0.017). Such marked reduction in plasma CETP activity resulted primarily from a net decrease in the rate of transfer of CE from HDL to triglyceride-rich lipoproteins (-31%; P = 0.03) and more specifically to VLDL-1 (-44%; P = 0.02) and VLDL-2 (-29%; P < 0.03), whereas rates of CE transfer from HDL to IDL were not significantly modified by drug therapy. When the rate of CE transfer from HDL to apoB-containing lipoproteins is expressed relative to plasma lipoprotein mass concentration, the relative capacity of each triglyceride-rich lipoprotein subfraction to accept CEs from HDL can be estimated. In type IIB subjects, IDL particles displayed a significantly higher capacity to accept CEs from HDL (10.6 ± 0.9 µg CE transferred/h·mg lipoprotein mass) in comparison with that of VLDL-1 (7.6 ± 0.9 µg CE transferred/h·mg lipoprotein mass; P = 0.04) and VLDL-2 (7.2 ± 0.8 µg CE transferred/h·mg lipoprotein mass; P = 0.01). Finally, ciprofibrate therapy did not modify the relative capacity of triglyceride-rich lipoprotein subfractions to accept CEs from HDL, suggesting that fibrate-mediated reduction in CE transfer occurs as a result of a decrease in lipoprotein particle acceptor number rather than as a change in CE acceptor capacity per particle.
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To determine whether ciprofibrate-mediated modifications in the quantitative and qualitative profile of plasma lipoprotein particles might influence cellular free cholesterol efflux, we measured the capacity of whole plasma from each type IIB patient before and after fibrate therapy to mediate cholesterol efflux from cultured rat Fu5AH hepatoma cells expressing high levels of SR-BI (23). Using 20-fold diluted plasma in triplicate assays, a significant elevation (+13%; P = 0.01) in the capacity of plasma from type IIB subjects to mediate free cholesterol efflux from hepatoma cells was observed after ciprofibrate treatment (Fig. 4A). In parallel, we determined the capacity of the apoB-containing lipoprotein-deficient d > 1.063 g/ml fraction, isolated from type IIB plasmas before and after fibrate therapy to mediate cholesterol efflux from Fu5AH cells. Interestingly, the d > 1.063 g/ml fraction isolated from plasmas of fibrate-treated type IIB subjects displayed a significantly higher capacity (+22%; P = 0.01) compared with baseline to mediate cholesterol efflux from rat hepatoma cells. In addition, the d > 1.125 g/ml fraction (from which both apoB-containing lipoproteins and HDL-2 particles had been removed) isolated from plasmas of fibrate-treated type IIB subjects displayed a significantly higher efflux capacity (+16%; P = 0.006) compared with those isolated from plasmas of nontreated patients. Therefore, part of the increased efflux capacity of the HDL-3-containing d > 1.063 g/ml fraction is associated with the d > 1.125 g/ml fraction.
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, using 20-fold or 40-fold diluted plasma in triplicate assays, plasma-mediated free cholesterol efflux from ABCA1-expressing J774 mouse macrophage cells was not significantly modified by fibrate therapy. Pretreatment of the cells with cAMP markedly increased cholesterol efflux to plasma (at least +40%); nonetheless, no significant changes in the capacity of plasma from type IIB subjects to mediate free cholesterol efflux via the ABCA1 transporter was observed after ciprofibrate treatment. |
Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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agonist, has been shown not only to induce preferential reduction in atherogenic apoB-containing lipoprotein subclasses (i.e. large VLDL-1, small VLDL-2, and dense LDL subspecies) and in CETP-mediated CE transfer from HDL to VLDL-1 and VLDL-2 subfractions in type IIB hyperlipoproteinemia, but also to induce significant elevation in antiatherogenic HDL-3 levels; this effect occurred concomitantly with an increase in cellular free cholesterol efflux to the HDL-3 subfraction (d > 1.125 g/ml) mediated by the SR-BI receptor in rat hepatoma cells. Our findings on ciprofibrate action are consistent with those previously reported for reduction in plasma cholesterol and triglyceride levels (-20% and -45%, respectively) in atherogenic hyperlipoproteinemia (28, 29). Equally, triglyceride-rich lipoprotein (VLDL-1 + VLDL-2 + IDL) levels were significantly reduced (-25%) on drug administration. Such reduction primarily involved major decrements in both VLDL-1 (-40%) and VLDL-2 (-25%) subfractions, whereas no significant effect of ciprofibrate was detected on plasma levels of IDL particles. Such a differential impact of ciprofibrate on individual triglyceride-rich lipoprotein subspecies has been previously observed with other potent fibrates (12, 13). Several studies, including the prospective Québec Cardiovascular Study (30), have demonstrated that elevated levels of small, dense LDL (>100 mg/dl) are intimately associated with elevated risk for the development of premature coronary artery disease. In the present study, the LDL subclass profile, in which small dense LDL predominated at baseline, was progressively normalized by ciprofibrate toward an intermediate pattern in which the peak corresponded to a predominance of large, buoyant LDL particles. Ciprofibrate qualitatively normalized the atherogenic, dense LDL profile characteristic of the IIB phenotype by specifically reducing plasma levels of dense LDL subfractions (LDL-4 and LDL-5, -46%) and increasing those of intermediate LDL subspecies (LDL-3, +27%) of high receptor affinity (11). These findings are entirely consistent with previous observations of the impact of ciprofibrate therapy on LDL particle profile (9).
Despite a slight but significant increase in total HDL mass (+13%; P = 0.05) as determined by chemical analysis, plasma HDL-cholesterol and apoAI levels were not significantly modified by ciprofibrate but showed a trend toward elevation (+14% and +5%, respectively; Table 1). This observation was accounted for by fibrate-induced modification in HDL subspecies profile in which levels of individual cholesterol-poor HDL-3 subfractions (HDL-3a, HDL-3b, and HDL-3c) were significantly increased (+13%, +22%, and +48%, respectively), whereas no effect of ciprofibrate was detected on CE-rich, large HDL-2 subfraction levels (Fig. 3). The impact of ciprofibrate on HDL particle profile presently observed in type IIB hyperlipoproteinemia is in agreement with those previously described for two other fibric acid derivatives, bezafibrate and gemfibrozil (12). The elevation in plasma HDL level typically observed during fibrate therapy has been frequently attributed to LPL activity, a key determinant of plasma HDL concentration, as a result of the sequestration of surface fragments from triglyceride-rich lipoprotein to the HDL pool (31). Indeed, fibrates can induce hepatic LPL gene expression through the activation of PPAR- (32). In agreement with a previous report (33), ciprofibrate induced elevation in postheparin LPL activity by approximately 20% (data not shown). In addition, we observed a significant reduction (-25%) in plasma CETP-mediated CE transfer from HDL to apoB-containing lipoproteins. It is well established that CETP is actively involved in the intravascular remodeling of HDL particles. Indeed, under certain conditions, CETP can induce the formation of small HDL particles at the expense of larger HDL (34). The ciprofibrate-induced reduction in plasma CETP activity might therefore be associated with a decrease in circulating levels of small HDL particles. However, because there is an inverse relationship between HDL-2 levels and HL activity (35), both LPL and HL might have opposing effects to CETP on plasma HDL concentration and particle profile. Thus, it has been demonstrated that HL can convert large triglyceride-rich HDL into smaller particles (36). Moreover, elevation in HL activity after ciprofibrate treatment has been reported (33), and a tendency to such an increase in HL activity was detected in the present study in the IIB phenotype (data not shown). Thus, the preferential increase in plasma HDL-3 subspecies might result from the combined action of both LPL and HL. These findings strongly suggest that the ciprofibrate-induced elevation in HL and LPL activity may counterbalance the drug-induced reduction of CETP activity on the intravascular remodeling of HDL particles.
To determine whether ciprofibrate-induced modification in the quantitative and qualitative features of plasma lipoprotein particles may influence their capacity to act as acceptors of cellular free cholesterol, we evaluated the capacity of type IIB hyperlipidemic plasma before and after ciprofibrate therapy to induce cholesterol efflux from Fu5AH rat hepatoma cells, which express high levels of the SR-BI receptor (24). Indeed, the SR-BI receptor pathway represents a critical component of the reverse cholesterol transport process, because this receptor is expressed at high levels in human monocyte-macrophages and lipid-loaded foam cells; indeed, the latter are key cellular components of lipid-rich, atherosclerotic plaques (18). Using the Fu5AH cell system in which cholesterol efflux occurs primarily via SR-BI receptors (37), we observed a significant increase (+13%; P = 0.01) in the capacity of whole plasma from ciprofibrate-treated patients to remove cellular free cholesterol compared with the plasma of type IIB patients before treatment. Because the cellular cholesterol efflux capacity of plasma in the Fu5AH cell system is known to be highly sensitive to HDL-phospholipid concentrations (38), the elevated cholesterol efflux capacity of posttreatment plasma can be related to the fibrate-induced increase in HDL-3 levels. Indeed, the total HDL-3 fraction isolated from the plasma of ciprofibrate-treated patients displayed an elevated capacity (+16%) to mediate free cholesterol efflux compared with HDL-3 isolated from plasma of subjects at baseline (Fig. 4A). Various cell lines, such as J774 mouse macrophages, display an enhanced cholesterol efflux capacity to lipid-free/lipid-poor apolipoproteins, including apoAI, apoAIV, and apoE, when stimulated with cAMP (39). Pretreatment of J774 cells by cAMP enhances the expression of the ABCA1, and thus up-regulation of ABCA1 gene expression is associated with increase in lipid-poor apolipoprotein-mediated efflux of cholesterol. In the present study, we demonstrated that free cholesterol efflux mediated by plasma from type IIB patients before and after fibrate therapy was not influenced by up-regulated expression of the ABCA1 transporter, suggesting that ciprofibrate did not influence plasma levels of lipid-free/lipid-poor apolipoproteins. In addition and in contrast to previous reports (28), we did not observe a significant elevation in plasma apoAI levels after ciprofibrate therapy. This finding contrasts with studies on the regulation of hepatic apoAI gene expression, carried out in human apoAI transgenic mice, which demonstrated an induction of human apoAI by fibrates (40). Taken together, these observations strongly suggest that HDL particles, mainly HDL-3 subspecies, that are active in facilitating cholesterol efflux are preferentially formed and/or accumulate in type IIB hyperlipidemic plasma after ciprofibrate therapy, possibly as a result of the concerted actions of LPL, HL, and CETP, rather than as a result of an increase in the hepatic production of nascent apoAI-containing HDL particles or from intravascular remodeling of preexisting HDL particles. Because both SR-BI/-1 and ABCA1 transporter gene expression in human macrophages is induced by fibrate-activated PPAR- (32), we cannot rule out the possibility that ciprofibrate therapy might stimulate cellular free cholesterol efflux from macrophages by this pathway in vivo.
The integrated action of ciprofibrate in atherogenic type IIB dyslipidemia is summarized in Fig. 5. Marked reduction in circulating levels (and particle numbers) of VLDL-1 and VLDL-2 of hepatic origin leads to correction of the dense LDL profile, with normalization of the intravascular remodeling cascade of apoB-containing particles (4, 5, 6, 13); reduced CETP-mediated CE transfer from HDL and increased LPL activity are integral features of such effects. Equally, preferential elevation in dense, CE-poor HDL-3 particle subclasses occurs as a result of increased LPL activity on triglyceride-rich particles and of increased hepatic production of apoAI and apoAII; such particles enhance SR-BI-mediated cellular cholesterol efflux and, as a consequence, the reverse transport of cholesterol to the liver for excretion. Considered together, these actions of ciprofibrate contribute significantly to reduction in the atherosclerotic burden—and thus cardiovascular risk—in atherogenic type IIB hyperlipoproteinemia.
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Acknowledgments |
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Footnotes |
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Abbreviations: ABCA1, ATP-binding cassette transporter-1; apo, apolipoprotein; CE, cholesteryl ester; CETP, CE transfer protein; FBS, fetal bovine serum; HDL, high-density lipoprotein(s); HL, hepatic lipase; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein(s); LPL, lipoprotein lipase; PPAR, peroxisome proliferator-activated receptor; SR-BI, scavenger receptor class B, type I; VLDL, very low-density lipoprotein(s).
Received February 6, 2003.
Accepted April 14, 2003.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) a 6-wk period of ciprofibrate (100 mg/d) treatment. Values are means ± SEM. *, P < 0.05; and ***, P < 0.0005 vs. before treatment.
) and after (
) a 6-wk period of ciprofibrate treatment (100 mg/d). Values are means ± SEM. *, P < 0.05 vs. before treatment.
