Fenofibrate improves the atherogenic lipid profile and enhances LDL resistance to oxidation in HIV-positive adults

Atherosclerosis 172 (2004) 273–279

Fenofibrate improves the atherogenic lipid profile and enhances LDL resistance to oxidation in HIV-positive adults Stephanie Badiou a , Corinne Merle De Boever b , Anne-Marie Dupuy a , Vincent Baillat b , Jean-Paul Cristol a,∗ , Jacques Reynes b a

Laboratoire de Biochimie des Lipides, Department of Biochemistry, 371 av du doyen Gaston Giraud, Hˆopital Lapeyronie, CHU Montpellier F34295, France b Department of Infectious Diseases, CHU Montpellier F34295, France Received 6 June 2003; received in revised form 12 September 2003; accepted 16 October 2003

Abstract Background: Low HDL-cholesterol, hypertriglyceridemia (HTG) and occurrence of small dense LDL could be involved in increased cardiovascular risk in HIV-infected patients. This study evaluates the effects of fenofibrate and/or Vitamin E on lipoprotein profile. Design: Thirty-six HIV-positive adults with fasting triglycerides (TGs) ≥2 mmol/l and stable antiretroviral therapy (ART) were randomly assigned to receive either micronised fenofibrate (200 mg/day) or Vitamin E (500 mg/day) for a first period of 3 months and the association of both for an additional 3-month period. Methods and results: Total cholesterol, HDL-C, LDL-C, triglycerides, apoA1, apoB, apoCIII, lipoprotein composition, LDL size and LDL resistance to copper-induced oxidation were determined before initiation of fenofibrate or Vitamin E, and 3 and 6 months thereafter. Three months of fenofibrate treatment results in a significant decrease in triglycerides (−40%), apoCIII (−21%), total cholesterol (−14%), apoB (−17%) levels, non-HDL-C (−17%), TG/apoA1 ratio in HDL (−27%) associated with an increase in HDL-C (+15%) and apoA1 (+11%) levels. Moreover, fenofibrate increases LDL size and enhances LDL resistance to oxidation. Three months of Vitamin E supplementation only improves LDL resistance to oxidation and addition to fenofibrate results in a slightly greater effect. Conclusion: Fenofibrate therapy improves the atherogenic lipid profile in HIV-positive adults with hypertriglyceridemia. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Dyslipidemia; Fenofibrate; HDL; HIV; LDL oxidation; LDL size; Triglycerides

1. Introduction The emergence of HIV infection as a potential risk factor for coronary heart disease (CHD) has been demonstrated in several studies [1–3]. Beyond classical risk factors such as tobacco, insulin resistance, and inflammation, specific HIV-related dyslipidemia could be involved in atherogenesis. Hypertriglyceridemia (HTG) is a frequent abnormality observed in HIV-positive patients receiving a protease inhibitor [4], although it was commonly seen before initiation of antiretroviral therapy (ART) [5]. This hypertriglyceridemic state leads to changes in cholesterol ester (CE) and triglyceride (TG) exchange pathways between lipoparticles, resulting in decreased HDL particles and occurrence of small ∗ Corresponding author. Tel.: +33-4-67-33-83-15; fax: +33-4-67-33-83-93. E-mail address: [email protected] (J.-P. Cristol).

dense LDL (sdLDL) [6], highly prevalent in HIV-infected patients [7,8]. These sdLDL, particularly susceptible to in vitro oxidation [9] and more able to infiltrate the subendothelial space, are a predictive factor for CHD [10]. Low HDL-C level, a common feature of HIV infection [5] is an additional independent cardiovascular risk factor [11] since beyond reverse cholesterol transport, HDL particles exhibit antioxidant, anti-inflammatory, and anticoagulant properties [12]. The association of sdLDL, susceptible to oxidation, with decreased HDL, a protective factor against LDL oxidation, leads to an increase in oxidatively modified atherogenic lipoparticles. Thus, management of dyslipidemia and prevention of oxidative stress appears as an option to reduce a potential increased CHD risk in HIV-infected patients. In such pathological conditions previously observed in non-insulin-dependent diabetes mellitus, two therapeutic strategies have been proposed. On the one hand, fibrate therapy that decreases serum TG, raises HDL-C, and

0021-9150/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2003.10.006

274

S. Badiou et al. / Atherosclerosis 172 (2004) 273–279

normalizes the atherogenic LDL profile [13]; on the other hand, Vitamin E supplementation that increases LDL resistance to ex vivo oxidation [14] and prevents oxidative stress [15]. The aim of this study was to evaluate the effects of fenofibrate and/or Vitamin E in HIV-infected patients with hypertriglyceridemia. Efficacy was assessed mainly by changes in TG and HDL-C levels, in LDL size and LDL oxidability.

months. In a second step all patients were receiving both fenofibrate (200 mg/daily) and Vitamin E (500 mg/daily) for an additional 3-month period. Biological parameters were performed on venous blood samples collected after an overnight fast, before initiation of treatment, and 3 and 6 months thereafter. There was no change in dietary habits throughout the study.

2. Material and methods 2.1. Study design Forty HIV-infected adults with fasting triglyceride levels ≥2 mmol/l and, for at least 4 months, stable antiretroviral therapy (CD4 cell count >100 mm−3 , HIV-1 viral load <5000 copies/ml) were enrolled in this study through the department of Infectious Diseases, University Hospital of Montpellier, France. Antiretroviral drugs used are listed in Table 1. The patients were assigned to the different clinical stages (A, B and C) of HIV infection according to the 1993 Classification of the Centers for Diseases Control and Prevention [16]. Presence of lipodystrophy was determined by physical examination on the basis of peripheral lipoatrophy and/or central lipodystrophy. Measurement of blood pressure was performed at baseline and hypertension was defined as systolic/diastolic pressure >140/90 mmHg. Exclusion criteria were pregnant women, presence of diabetes, fasting glycemia >7 mmol/l, hepatic failure, renal function impairment (creatininemia >150 ␮mol/l), allergy to fibrate, anti-Vitamin K therapy and use of hypolipemiant and/or antioxidant drugs 3 months before inclusion. Patients were randomized 1:1 to receive either micronised fenofibrate: group First-feno (LIPANTHYL® 200 mg, once daily) or Vitamin E: group First-toco (alpha-tocopherol, TOCOPHAN® 500 mg once daily) for a first period of 3 Table 1 Antiretroviral drugs used (%) for all patients (n = 36) and in groups First-feno (n = 18) and First-toco (n = 18)

Lamivudine Didanosine Abacavir Zidovudine Ritonavir Lopinavir/ritonavir Efavirenz Nevirapine Stavudine Nelfinavir Indinavir Amprenavir Saquinavir Tenofosvir

All patients

First-feno

First-toco

74 43 31 31 29 29 26 23 20 14 14 9 5 5

71 47 18 35 35 18 24 18 29 12 24 12 0 0

78 39 44 28 22 39 28 28 11 17 6 6 11 11

Subjects withdrew from the study for any of the following reasons: appearance of an exclusion criteria, a virological failure, an antiretroviral therapy modification, adverse events of fibrate therapy, increase in transaminases greater than threefold from baseline’s values or CPK higher than fivefold normal values. This study was approved by The Ethics Committee of Montpellier and all patients signed informed consent. 2.2. Laboratory methods Routine biochemical parameters were measured using an Olympus 2700 analyzer (Olympus, France). Insulin levels were determined with a radioimmunoassay and HOMA index was calculated with the formula: insulin (␮UI/l) × glucose (mmol/l)/22.5 [17]. High sensitive C-Reactive Protein (hs-CRP) levels were determined using an immunoturbidimetric method on a Olympus 2700 analyzer. Total cholesterol (TC), HDL-C, TG, and phospholipid (PL) levels were measured in serum by routine enzymatic methods (Konepro, Konelab). Non-HDL-C was estimated as (TC–HDL-C) and LDL-C was calculated by the Friedwald’s formula for patients with TG ≤ 4.5 mmol/l. Apolipoprotein (apo) A1, apoB and apoCIII concentrations were determined by immunonephelometric assay (Behring Nephelemeter 100, Behring Diagnostic SA). Within-run and between-run imprecisions for apolipoprotein determinations were less than 3%. The size of the predominant LDL subfraction was determined by plasma electrophoretic migration in nondenaturant polyacrylamide gradient gels (Spiragel 1.5–25%, Lara-Spiral), according to the procedure described by Blanche et al. [18] using markers of known diameter (Amersham Pharmacia calibration kit including thyroglobulin, ferritin, catalase and Duke Scientific Corp 38.0 nm latex beads). Intra-assay imprecision was <1%. Electrophoresis time was 1 h at 70 V then 24 h at 150 V. Gels were stained with Coomassie brillant blue G250 for 1 h and destained for 12 h, then scanned using an optical densitometric image analyzer (Fisher Bioblock Scientific). Diameter of the major LDL peak was calculated with the Bio-1D software (Fisher Bioblock Scientific) and small dense LDL was defined as <25.5 nm.

S. Badiou et al. / Atherosclerosis 172 (2004) 273–279

Lipoproteins were prepared by sequential ultracentrifugation from 5 ml of plasma (Kontron TGA-50, Kontron). Plasma density was adjusted with potassium bromide to 1.019 g/ml to obtain VLDL/IDL fractions after ultracentrifugation at 40,000 rpm for 20 h. After removing the upper layer, infranatant density was adjusted to 1.063 g/ml to obtain LDL fraction (40,000 rpm for 24 h). HDL particles were obtain at the density 1.215 g/ml (45,000 rpm for 24 h). Levels of TC, TG, PL and apolipoproteins were determined by the same procedure as in serum. Lipoprotein composition was analyzed using the TC/apo, TG/apo and PL/apo ratios. Vitamin E (alpha-tocopherol) concentrations in serum and LDL were measured by high performance liquid chromatography, as previously described [19]. For serum, results were normalized and expressed as vitE/total lipids (␮mol/mmol ratio). For LDL, results were expressed as the molar vitE/apoB ratio. LDL oxidation was initiated with copper (Cu(II), 5 ␮M) and continuously monitored during 6 h by measuring the increase in absorbance at 234 nm due to conjugated diene formation, with a spectrophotometer UVIKON 930 (Kontron Instruments). The absorption data were analyzed by the NELOP software [20] in order to determine (i) the lag-time corresponding to consumption of the endogenous antioxidants, mainly Vitamin E, (ii) the maximal propagation rate (Vmax ) of lipid peroxidation given by the peak of the first derivative of the absorption curve, (iii) the time of half oxidation (T1/2 ) and (iv) the slope of the propagation phase. 2.3. Statistical analysis Results were expressed as mean ± S.E.M. Categorical data were compared using the χ2 -test. Log10 transformed values for TG and for CRP were performed prior to carry out statistical analysis. A paired t-test was used for repeated value comparison. Between-group difference was performed with an unpaired Student’s t-test. Statistical significance was assumed for P < 0.05.

275

3. Results 3.1. Characteristics of patients Four patients, out of the forty subjects screened, withdrew from the study prior to randomization. One owing to high TG levels (33 mmol/l) considered as a life threatening event, two because of a glycemia >7 mmol/l and one because of antiretroviral therapy modification. The 36 remaining patients were: 18 randomly assigned to the group First-feno, receiving first only fenofibrate, and eighteen assigned to the group First-toco, receiving first only Vitamin E. The characteristics of the patients are presented in Table 2, there was no statistical difference between the two groups. No patient had insulin resistance, defined as HOMA index greater than 4 (mean HOMA:1.3 ± 0.2). Among the 22 patients suffering from lipodystrophy, 7 presented with a mixed syndrome, 3 had isolated central lipodystrophy and 12 had isolated peripheral lipoatrophy. All patients were antiretroviral treatment-experienced and there was no significant change in viral load or CD4 cell counts throughout the study period. No patient had acute opportunistic infection. Few events were observed: upper respiratory infections (n = 10), pneumonia (n = 2), pruritus (n = 2), depression (n = 2), insomnia (n = 1), diminished libido (n = 1), Fanconi syndrome related to tenofovir (n = 1), gastroenteritis (n = 1), abdominal pain and diarrhea secondary to lopinavir/ritonavir (n = 1) and allergic shock of unknown origin (n = 1). We noted five cases of elevated CPK (grade II toxicity), of which three (one at M3, two at M6) occurred in the First-feno group and two (one at M3, one at M6) in the First-toco group. No adverse event led to study discontinuation or dose modification. 3.2. Baseline state (M0) When compared to the laboratory reference range, the entire group of HIV enrolled patients exhibit: (i) a moderate increase in TC (6.64±0.23 mmol/l), LDL-C (4.08 ± 0.20 mmol/l) and apoB (1.41 ± 0.05 g/l) levels;

Table 2 Characteristics of patients at baseline

Age (years) Body mass index (kg/m2 ) Gender (M, %) Hypertension (%) Current smoker (%) Lipodystrophy (%) Family history of CHD (%) Clinical stage A/B/C (%) Exposure to ART (months) Exposure to PI (months) CD4 cell counts (mm−3 ) Viral load (copies/ml)

All patients (n = 36)

First-feno (n = 18)

First-toco (n = 18)

44 ± 2 23 ± 1 86 8 52 61 21 36/39/25 51 ± 4 30 ± 3 476 ± 35 135 ± 59

46 ± 3 24 ± 1 78 5 53 72 18 44/28/28 51 ± 7 31 ± 5 530 ± 48 94 ± 46

43 ± 2 23 ± 1 94 11 50 50 25 28/50/22 52 ± 7 29 ± 5 421 ± 48 177 ± 110

CHD: coronary heart disease, ART: antiretroviral therapy, PI: protease inhibitors.

276

S. Badiou et al. / Atherosclerosis 172 (2004) 273–279

(ii) a substantial increase in TG (3.48 ± 0.32 mmol/l), in non-HDL-C (5.53 ± 0.20 mmol/l) and in apoCIII (0.15 ± 0.01 g/l) levels associated with a decreased LDL size (24.92 ± 0.08 nm) and a 86% prevalence of sdLDL; (iii) decreased HDL-C (1.01 ± 0.04 mmol/l) and apoA1 (1.25 ± 0.03 g/l) levels associated with a TG overload in HDL fraction (TG/apoA1 molar ratio: 5.65 ± 0.3); (iv) increased hs-CRP levels (4.12 ± 0.75 mg/l). Detailed mean values in groups First-feno and First-toco are listed in Table 3. All parameters are statistically similar between both groups, except higher LDL-C levels in the First-feno group (P = 0.02) associated with a trend towards hypercholesterolemia (P = 0.08). 3.3. Evaluation at 3 months (M3) 3.3.1. First-feno group Fenofibrate treatment leads to a significant reduction of TG from 3.49 to 2.09 mmol/l (−40%), of apoCIII (−21%),

of apoB (−17%), of non-HDL-C (−17%), of TC (−14%), and LDL-C (−14%) levels whilst HDL-C and apoA1 increase (+15 and +11%, respectively). Moreover, a significant increase in mean LDL size from 24.88 to 25.30 nm (P < 0.005) is observed and eight patients shift from a pattern B (<25.5 nm) to a pattern A (≥25.5 nm). A significant decrease in TG content of HDL fraction is evidenced by a decreased TG/apoA1 molar ratio (−27%, P < 0.001). No change of CRP levels are observed during this period (Table 3). 3.3.2. First-toco group Vitamin E supplementation results in a 50% increase in serum vitE/lipids (␮mol/mmol) ratio associated with a 36% increase in vitE/apoB molar ratio in LDL (Table 3). No significant difference is observed on lipid parameters, lipoprotein composition, LDL size or CRP levels (Table 3). In both groups an enhanced resistance to ex vivo oxidation is evidenced when compared to baseline. This protective effect delayed the oxidative process as shown by an increased lag-time duration (+25 and +20% in First-feno

Table 3 Serum and lipoproteins parameters, expressed as mean ± S.E.M., at baseline (M0), 3 months (M3) and 6 months (M6) thereafter Group

M0

M3

M6 0.20∗

TG (mmol/l)

F-feno F-toco

3.49 ± 0.56 3.46 ± 0.35

2.09 ± 3.47 ± 0.45

2.04 ± 0.21∗ 2.16 ± 0.19∗,§

TC (mmol/l)

F-feno F-toco

7.04 ± 0.31 6.24 ± 0.32

6.04 ± 0.31∗ 6.34 ± 0.29

6.07 ± 0.25∗ 5.81 ± 0.25

LDL-C (mmol/l)

F-feno F-toco

4.70 ± 0.23 3.53 ± 0.26

3.94 ± 0.24∗ 3.84 ± 0.25

3.92 ± 0.19∗ 3.66 ± 0.20

HDL-C (mmol/l)

F-feno F-toco

1.00 ± 0.06 1.02 ± 0.05

1.16 ± 0.07∗ 1.06 ± 0.04

1.13 ± 0.07∗ 1.17 ± 0.05∗,§

Non-HDL-C (mmol/l)

F-feno F-toco

5.87 ± 0.24 5.22 ± 0.31

4.89 ± 0.31∗ 5.24 ± 0.29

4.93 ± 0.27∗ 4.64 ± 0.26∗,§

apoA1 (g/l)

F-feno F-toco

1.21 ± 0.05 1.28 ± 0.04

1.34 ± 0.08∗ 1.33 ± 0.04

1.37 ± 0.07∗ 1.42 ± 0.04∗,§

apoB (g/l)

F-feno F-toco

1.53 ± 0.07◦ 1.28 ± 0.07

1.28 ± 0.08∗ 1.36 ± 0.09

1.33 ± 0.07∗ 1.19 ± 0.07∗,§

apoCIII (g/l)

F-feno F-toco

0.15 ± 0.01 0.14 ± 0.01

0.12 ± 0.01∗ 0.15 ± 0.01

0.11 ± 0.01∗ 0.11 ± 0.01∗,§

LDL size (nm)

F-feno F-toco

24.88 ± 0.12 24.96 ± 0.11

25.30 ± 0.11∗ 24.87 ± 0.15

25.42 ± 0.12∗ 25.33 ± 0.14∗,§

TG/apoA1 in HDL

F-feno F-toco

5.68 ± 0.53 5.62 ± 0.28

4.15 ± 0.31∗ 5.68 ± 0.48

4.31 ± 0.32∗ 4.56 ± 0.31∗,§

vitE/lipids in serum

F-feno F-toco

3.26 ± 0.21 3.22 ± 0.21

3.31 ± 0.18 4.82 ± 0.33∗

5.10 ± 0.41∗,§ 4.99 ± 0.39∗

vitE/apoB in LDL

F-feno F-toco

6.42 ± 0.47 6.86 ± 0.47

6.29 ± 0.54 9.44 ± 0.79

9.41 ± 0.88∗ 10.03 ± 0.98∗,§

CRP (mg/l)

F-feno F-toco

4.80 ± 1.25 3.39 ± 0.82

4.71 ± 1.61 3.01 ± 0.62

F-feno: First-feno group, n = 18; F-toco: First-toco group, n = 18. ∗ P < 0.05: comparison M3/M0, M6/M0. § P < 0.05: comparison M6/M3.

5.89 ± 1.75 2.71 ± 0.61

S. Badiou et al. / Atherosclerosis 172 (2004) 273–279

277

Table 4 Parameters of LDL resistance to ex vivo copper-induced oxidation, expressed as mean ± S.E.M., at baseline (M0), 3 months (M3) and 6 months (M6) thereafter Group

M0

M3

M6 5∗

Lag-time (min)

F-feno F-toco

61 ± 3 61 ± 3

76 ± 73 ± 3∗

87 ± 6∗,§ 75 ± 4∗

Time of half oxidation (min)

F-feno F-toco

84 ± 3 81 ± 3

98 ± 5∗ 94 ± 3∗

112 ± 7∗,§ 97 ± 4∗

Vmax

F-feno F-toco

0.022 ± 0.001 0.023 ± 0.001

0.020 ± 0.001∗ 0.021 ± 0.001∗

0.019 ± 0.001∗ 0.019 ± 0.001∗,§

Slope

F-feno F-toco

0.020 ± 0.001 0.022 ± 0.001

0.019 ± 0.001∗ 0.020 ± 0.001∗

0.018 ± 0.001∗ 0.018 ± 0.001∗,§

F-feno: First-feno group, n = 18; F-toco: First-toco group, n = 18. ∗ P < 0.05: comparison M3/M0, M6/M0. § P < 0.05: comparison M6/M3.

and First-toco groups, respectively) and increased time of half oxidation (+17 and +16% in First-feno and First-toco groups, respectively). Additionally, significant decreases in maximal propagation rate and in the slope of propagation phase are observed (Table 4). 3.4. Evaluation at 6 months (M6) In the First-feno group, serum lipid parameters, lipoprotein composition, LDL size and CRP levels are similar to M3 state (Table 3). Addition of Vitamin E to fenofibrate results to expected increases in serum vitE/lipids ratio (+54%) and in vit E/apoB ratio in LDL (+49%). Furthermore, LDL resistance to ex vivo oxidation, previously enhanced by fenofibrate, is more pronounced as demonstrated by a further significant increase in lag-time (+14%) and time of half oxidation (+15%) versus M3 (Table 4). In the First-toco group, administration of fenofibrate after Vitamin E supplementation results in significant decreases of TG from 3.47 to 2.16 mmol/l (−38%), of apoCIII (−26%), of apoB (−12%) of non-HDL-C (−11%) associated with a rise in HDL-C (+10%) and apoA1 (+7%) levels when compared to the 3 months values. Decrease in TC (−8%) and LDL-C (−5%) did not reached statistical significance (Table 3). LDL size increases from 24.87 to 25.33 nm (P < 0.001) and nine patients change from a pattern B to a pattern A. Moreover, a significant decreased TG/apoA1 molar ratio in HDL fraction is observed (−20%, P < 0.001). No effect appears on CRP levels (Table 3). Lag-time and time of half oxidation values are not statistically different from M3 values. Significant decreases in maximal propagation rate and in slope of propagation phase versus M3 state are observed (Table 4). Considering all patients, the comparison between M0 and M6 demonstrate (i) a significant reduction of serum TG by 40%, of apoCIII by 26%, of non-HDL-C by 13%, of apoB by 10%, of TC by 10% associated with an increase in HDL-C by 14%, in apoA1 by 12%, in LDL size by 2%; (ii) signif-

icant increases in serum vitE/lipids (+56%) and vitE/apoB in LDL (+46%) associated with a protective effect on LDL oxidation evidenced by an increased lag-time (+33%), an increased time of half oxidation (+26%) and reduced maximal propagation rate (−15%) and slope (−16%). Moreover, the decrease in serum TG levels is associated with a significant decrease in TG content of HDL (TG/apoA1 reduced by 22%). Seventeen patients (45%) return to serum TG levels lower than 2 mmol/l and prevalence of TG >4 mmol/l decreases from 29 to 5%. Presence of sdLDL decreases from 86 to 47% and prevalence of HDL-C <1.15 mmol/l decreases from 70 to 46%.

4. Discussion The introduction of potent combination antiretroviral therapy has significantly increased the survival rates of HIV-infected patients with improvement of life quality [21]. However, emergence of cardiovascular complications related to HIV infection per se and/or to ART have raised clinical concern [22,23]. The 5-year relative risk for cardiovascular disease was reported to be higher for HIV-infected patients receiving a protease inhibitor compared to HIV-negative subjects matched for sex and age [1]. Among the potential risk factors, dyslipidemia which was associated with an increased carotid intima media thickness [24] should be screened and managed. Recently, the DAD study reported a link between the prevalence of dyslipidemia and exposure of the patients to ART [2]. Since ART is defined to be used as a long-term treatment, complications such as ART-induced hypertriglyceridemia need to be managed. Our results show that fenofibrate significantly improves the atherogenic lipid profile of HIV-treated patients by a substantial reduction of TG associated with an increase in HDL-C, in LDL size and an enhanced resistance of LDL to ex vivo oxidation. The duration of lag-time is further enhanced when Vitamin E is added to fenofibrate, however

278

S. Badiou et al. / Atherosclerosis 172 (2004) 273–279

there is no additional effect if fenofibrate is used after Vitamin E. Only a few studies explored fibrate derivatives as a potential option in HIV-induced dyslipidemia management and without exploring the LDL size and/or the LDL oxidability. The 40% reduction in TG levels observed in our study is in agreement with previous report using bezafibrate (n = 25), gemfibrozil (n = 22) or fenofibrate (n = 22) [25]. Surprisingly, only a modest efficacy of gemfibrozil on TG and HDL-C levels in seventeen HIV-infected patients was observed by Miller et al. [26]. Efficacy of fenofibrate in severe hypertriglyceridemia (9 ± 1.4 mmol/l) was confirmed in twenty HIV-infected patients with a 53% reduction in TG levels and without any adverse side effects [27]. In our study, efficacy of fenofibrate on TG levels was demonstrated in moderate hypertriglyceridemia (3.5 ± 0.3 mmol/l). In agreement with previous results of Bonnet et al. [28], decrease in TG levels was associated with an important decrease in apoCIII serum levels (−26%), an endogenous inhibitor of lipoprotein lipase (LpL). Fibrate therapy in type II hyperlipidemia leads to a decrease in circulating VLDL, and especially in VLDL1 particles [29], known to favor the formation of sdLDL [6]. Since an alteration of LpL-mediated delipidation of VLDL1 was reported in HIV-infected patients [30], restored VLDL delipidation should decrease formation of sdLDL as previously reported in diabetic patients [13]. Indeed, the increase in LDL size observed after 3 months of fenofibrate therapy leads to a reduction of the enhanced prevalence of sdLDL (from 86 to 47%). Biochemical and metabolic features of sdLDL [31] including susceptibility to in vitro oxidation could account for sdLDL-related risk of CHD [10]. In our study, the enhanced LDL resistance to ex vivo oxidation observed with fenofibrate therapy could be related to the shift of sdLDL toward larger LDL [9]. In addition to the constant TG reduction and to the increase in LDL size, a significant decrease of total cholesterol and LDL-C was obtained in presence of high levels at baseline (group First-feno). Moreover, fenofibrate increased HDL-C by 14% and apoA1 levels by 12%, two protective factors for myocardial infarction [11,32]. This increase in HDL mass may be linked to the decrease in TG content. Indeed, cholesterol ester transfer protein (CETP) modulation by fibrates results in decreased TG content of HDL [33], as observed in our study, and may in turn slow the HDL clearance [34]. All these effects are consistent with the known peroxisome proliferated activated receptor alpha (PPAR␣) agonist effects of fibrates including apoCIII gene repression, LpL gene induction and modulation of the CETP activity [33,35]. Overall improvement in lipid profile with fenofibrate was not associated with a decrease in hs-CRP levels and inflammatory status as previously suggested in combined hyperlipidemia [36]. Although beneficial effects of Vitamin E in primary or/and secondary prevention of coronary heart disease remained

controversial [37,38], a Vitamin E supplementation could be carried out in diabetic type II patients in order to increase LDL resistance to ex vivo oxidation [14] and to improve oxidative disturbances [15]. Since an oxidative stress could occur in HIV disease [39], a Vitamin E supplementation may be proposed. In this study, Vitamin E significantly improves all parameters of LDL oxidation without any modification of serum lipid parameters or lipoprotein composition. Addition of Vitamin E after 3 months of fenofibrate treatment results in a further increased lag-time and time of half oxidation, whilst addition of fenofibrate after 3 months of Vitamin E results in a further decreased maximal propagation rate and slope. These results suggest an additional effect between fenofibrate and Vitamin E as previously reported in animal models [40]. However, Vitamin E increased only by 14% the duration of lag-time after fenofibrate treatment. Interest of Vitamin E in hypertriglyceridemic HIV-positive patients treated with fenofibrate is probably weak since no effect on lipid parameters can be expected and the maximal increase in lag-time duration is reached with fenofibrate (+25%). Considering the various cardiovascular risk factors in HIV-infected patients, management of dyslipidemia including decrease in TG, increase in HDL-C and in LDL size by fenofibrate appears as an effective option in order to reduce the potential risk of CHD.

Acknowledgements We are grateful to all patients and physicians for participating in this study. Moreover, we thank Christine Tramoni, Christine Delonca and the “Delegation de la Recherche Clinique du Centre Hospitalier Universitaire de Montpellier” for data monitoring and coordination of this study. Sponsorship: This work was supported by a grant of SIDACTION. Vitamin E and fenofibrate were provided by Arkopharma (France) and Fournier (France) laboratories, respectively.

References [1] Saves M, Chene G, Ducimetiere P, Leport C, Le Moal G, Amouyel P, et al. French WHO MONICA Project and the APROCO (ANRS EP11) Study Group. Risk factors for coronary heart disease in patients treated for human immunodeficiency virus infection compared with the general population. Clin Infect Dis 2003;37:292–8. [2] Friis-Moller N, Weber R, Reiss P, Thiebaut R, Kirk O, d’Arminio Monforte A, et al. DAD study group. Cardiovascular disease risk factors in HIV patients-association with antiretroviral therapy. Results from the DAD study. AIDS 2003;17:1179–93. [3] Currier JS, Taylor A, Boyd F, Dezii CM, Kawabata H, Burtcel B, et al. Coronary heart disease in HIV-infected individuals. J Acquir Immune Defic Syndr 2003;33:506–12. [4] Carr A, Samaras K, Burton S, Law M, Freund J, Chisholm DJ, et al. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 1998;12:F51–8. [5] Grunfeld C, Pang M, Doerrler W, Shigenaga JK, Jensen P, Feingold KR. Lipids, lipoproteins, triglyceride clearance, and cytokines in

S. Badiou et al. / Atherosclerosis 172 (2004) 273–279

[6] [7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

human immunodeficiency virus infection and the acquired immunodeficiency syndrome. J Clin Endocrinol Metab 1992;74:1045–52. Packard CJ, Shepherd J. Lipoprotein heterogeneity and apolipoprotein B metabolism. Arterioscler Thromb Vasc Biol 1997;17:3542–56. Stein JH, Klein MA, Bellehumeur JL, McBride PE, Wiebe DA, Otvos JD, et al. Use of human immunodeficiency virus-1 protease inhibitors is associated with atherogenic lipoprotein changes and endothelial dysfunction. Circulation 2001;104:257–62. Badiou S, Merle De Boever C, Dupuy AM, Baillat V, Cristol JP, Reynes J. Decrease in LDL size in HIV-positive adults before and after lopinavir/ritonavir-containing regimen: an index of atherogenicity? Atherosclerosis 2003;168:107–13. Chait A, Ronald L, Brazg M, Tribble DL. Susceptibility of small, dense, low-density lipoproteins to oxidative modification in subjects with the atherogenic lipoprotein phenotype, pattern B. Am J Med 1993;94:350–6. St-Pierre AC, Ruel IL, Cantin B, Dagenais GR, Bernard PM, Despres JP, et al. Comparison of various electrophoretic characteristics of LDL particles and their relationship to the risk of ischemic heart disease. Circulation 2001;104:2295–9. Despres JP, Lemieux I, Dagenais GR, Cantin B, Lamarche B. HDL-cholesterol as a marker of coronary heart disease risk: the Québec cardiovascular study. Atherosclerosis 2000;153:263–72. Nofer JR, Kehrel B, Fobker M, Levkau B, Assmann G, Von Eckardstein A. HDL and arteriosclerosis: beyond reverse cholesterol transport. Atherosclerosis 2002;161:1–16. Lemieux I, Laperrière L, Dzavik V, Tremblay G, Bourgeois J, Despres JP. A 16-week fenofibrate treatment increases LDL particle size in type IIA dyslipidemic patients. Atherosclerosis 2002;162:363–71. Reaven P, Herold D, Barnett J, Edelman S. Effects of Vitamin E on susceptibility of low density lipoprotein and low density lipoprotein subfractions to oxidation and on protein glycation in NIDDM. Diabetes Care 1995;18:807–16. Sharma A, Kharb S, Chugh SN, Kakkar R, Singh GP. Evaluation of oxidative stress before and after control of glycemia and after Vitamin E supplementation in diabetic patients. Metabolism 2000;49:160–2. 1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR 1992;41:1–19. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9. Blanche PJ, Gong EL, Forte TM, Nichols AV. Characterization of human high-density lipoproteins by gradient gel electrophoresis. Biochim Biophys Acta 1981;665:408–19. Cristol JP, Bosc JY, Badiou S, Leblanc M, Lorrho R, Descomps B, et al. Erythropoietin and oxidative stress in hemodialysis: beneficial effects of Vitamin E supplementation. Nephrol Dial Transpl 1997;12:2312–7. Morena M, Vaussenat F, Maggi MF, Canaud B, Cristol JP, Descomps B. Kinetic modeling of LDL oxidation using software (NELOP program) and validation in a population at vascular risk. J Soc Biol 1999;193:211–7. Palella Jr FJ, Delaney KM, Moorman AC, Loveless MO, Fuhrer J, Satten GA, et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV outpatient Study Investigators. N Engl J Med 1998;338:853–60. Holmberg SD, Moorman AC, Williamson JM, Tong TC, Ward DJ, Wood KC, et al. Protease inhibitors and cardiovascular outcomes in patients with HIV-1. Lancet 2002;360:747–8. Vittecoq D, Escaut L, Chironi G, Teicher E, Monsuez JJ, Andrejak M, et al. Coronary heart disease in HIV-infected patients in the highly active antiretroviral treatment era. AIDS 2003;17:S70–6.

279

[24] Seminari E, Pan A, Voltini G, Carnevale G, Maserati R, Minoli L, et al. Assessment of atherosclerosis using carotid ultrasonography in a cohort of HIV-positive patients treated with protease inhibitors. Atherosclerosis 2002;162:433–8. [25] Calza L, Manfredi R, Chiodo F. Statins and fibrates for the treatment of hyperlipidaemia in HIV-infected patients receiving HAART. AIDS 2003;17:851–9. [26] Miller J, Brown D, Amin J, Kent-Hughes J, Law M, Kaldor J, et al. A randomized, double-blind study of gemfibrozil for the treatment of protease inhibitor-associated hypertriglyceridaemia. AIDS 2002;16:2195–200. [27] Palacios R, Santos J, Gonzalez M, Ruiz J, Valdivielso P, Marquez M, et al. Efficacy and safety of fenofibrate for the treatment of hypertriglyceridemia associated with antiretroviral therapy. J Acquir Immune Defic Syndr 2002;3:251–3. [28] Bonnet E, Ruidavets JB, Tuech J, Ferrieres J, Collet X, Fauvel J, et al. Apoprotein c-III and E-containing lipoparticles are markedly increased in HIV-infected patients treated with protease inhibitors: association with the development of lipodystrophy. J Clin Endocrinol Metab 2001;86:296–302. [29] Milosavljevic D, Griglio S, Le Naour G, Chapman MJ. Preferential reduction of very low density lipoprotein-1 particle number by fenofibrate in type IIB hyperlipidemia: consequences for lipid accumulation in human monocyte-derived macrophages. Atherosclerosis 2001;155:251–60. [30] Schmitz M, Michl GM, Walli R, Bogner J, Bedynek A, Seidel D, et al. Alterations of apolipoprotein B metabolism in HIV-infected patients with antiretroviral combination therapy. J Acquir Immune Defic Syndr 2001;26:225–35. [31] Chapman J, Bruckert E. The atherogenic role of triglycerides and small dense low density lipoproteins: impact of ciprofibrate therapy. Atherosclerosis 1996;124(Suppl):21–8. [32] Walldius G, Jungner I, Holme I, Aastveit AH, Kolar W, Steiner E. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet 2001;358:2026–33. [33] Guerin M, Bruckert E, Dolphin PJ, Turpin G, Chapman MJ. Fenofibrate reduces plasma cholesteryl ester transfer from HDL to VLDL and normalizes the atherogenic, dense LDL profile in combined hyperlipidemia. Arterioscler Thromb Vasc Biol 1996;16:763–72. [34] Rashid S, Uffelman KD, Lewis GF. The mechanism of HDL lowering in hypertriglyceridemic, insulin-resistant states. J Diabetes Complications 2002;16:24–8. [35] Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart JC. Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation 1998;98:2088–93. [36] Malik J, Melenovsky V, Wichterle D, Haas T, Simek J, Ceska R, et al. Both fenofibrate and atorvastatin improve vascular reactivity in combined hyperlipidaemia (fenofibrate versus atorvastatin trial—FAT). Cardiovasc Res 2001;52:290–8. [37] Stephens NG, Parsons A, Schofield PM, Kelly F, Cheeseman K, Mitchinson MJ. Randomised controlled trial of Vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 1996;347:781–6. [38] Yusuf S, Dagenais G, Pogue J, Bosch J, Sleight P. Vitamin E supplementation and cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med 2000;342:154–60. [39] Elbim C, Pillet S, Prevost MH, Preira A, Girard PM, Rogine N, et al. The role of phagocytes in HIV-related oxidative stress. J Clin Virol 2001;20:99–109. [40] Chaput E, Maubrou-Sanchez D, Bellamy FD, Edgar AD. Fenofibrate protects lipoproteins from lipid peroxidation: synergistic interaction with alpha-tocopherol. Lipids 1999;34:497–502.