Effect of retinoids on LDL metabolism by macrophages

ARTICLE

Retinoids have been used in dermatological therapy for a long time, and it has also long been known that retinoids can cause unfavourable, atherogenic changes in serum lipids. They increase the concentration of very low density lipoprotein triglycerides (VLDL) and low density lipoprotein cholesterol (LDL) and decrease high density lipoprotein cholesterol (HDL). How side effects develop has not been clarified [1, 2].

Each cell and organ of the body is involved in lipid metabolism, with the serum lipid level being determined by the balance of absorption, synthesis, distribution and excretion. Hyperlipidemia is significant for its pathogenetic role in atherosclerosis. The monocyte-macrophage system with its important immunological function plays a key role in lipid metabolism, too [3-5]. Monocytes lose their specific LDL receptor, which is a characteristic of every cell, during the maturation to macrophages, and they then express scavenger receptors that are able to take up LDL in a modified form (oxidized, acetylated, malonated) [4]. Macrophages can mediate the oxidation of LDL [6].

LDL, entering by means of the specific receptor, does not cause cholesterol accumulation in the cell because intracellular cholesterol levels rapidly regulate LDL receptor levels and endogenous cholesterol synthesis [7]. Modified LDL uptake through the scavenger receptor is not so well regulated. Cholesterol entering the cell in this way may accumulate to form a so-called foam cell [8, 9]. Cholesterol loading of macrophages increases apolipoprotein E (apoE) secretion, the increase of apoE secretion being significant from the standpoint of lipid metabolism [10]. The presence of apoE facilitates the acquisition of cholesterol by HDL [11] and the excretion of cholesterol through the liver [12]. Increased apoE gene expression by macrophages in human atheroma may indicate this protective function [13].

Lipid metabolism in monocyte-derived macrophages (MDM) can be investigated in cell culture. Seventy two-hour cultures contain specific and scavenger receptors in approximately equal amounts. This makes it possible to perform parallel experiments to study the function of the two receptors [14]. In the present work we investigated the effect of retinoic acid on the LDL metabolism of macrophages derived from healthy volunteers and retinoid-treated patients in order to contribute to the explanation of the hyperlipidemia caused by retinoids.

Materials and methods

Preparation of monocyte-derived macrophage monolayers

Monocytes were separated from blood taken by venesection from 12 healthy volunteers with normolipemia and 5 males (average age: 19.1 years) suffering from cystic acne who were being treated with l mg/kg body weight Roaccutane^® (isotretinoin) for 2 months as described earlier [15]. Volunteers had given their informed consent. The cell suspension was cultured in RPMI 1640 medium (Gibco) supplemented with 5% fetal calf serum (Serva). Cells were cultured for 72 h in plastic trays with 12 wells (Flow) in an Assab CO₂ incubator under sterile conditions at 37° C. Each well contained about 5 x 10⁵ cells in 500 µl medium [5].

All trans retinoic acid (all-trans-RA) and 13-cis-retinoic acid (13-cis-RA) (a gift from Roche) were dissolved in ethanol and applied separately in a final concentration of 1 µM and incubated for 1-12 h. The same amount of ethanol was used in the medium of control experiments. Time course experiments showed a sharp decrease of apoE secretion after 8 h the rate of decrease being slower after 12 h.

Statistical analyses: T-test for independent samples was performed using Microsoft Exel 5.0 software.

Preparation of LDL

LDL was isolated from plasma of normolipidemic, healthy subjects by sequential density gradient ultracentrifugation [16]. The LDL concentration was expressed as µg protein/ml using the folin phenol reagent and the method of Lowry et al. [17]. LDL was acetylated by the repetitive addition of acetic anhydride [18]. Labelling of native and acLDL was performed by using ¹²⁵I (Amersham) [19]. The specific activity of the preparation was 300-400 cpm/ng.

Measurement of binding and intracellular degradation of ¹²⁵I-LDL and ¹²⁵I-acLDL

In order to determine the binding of the lipoproteins, ¹²⁵I-LDL and ¹²⁵I-acLDL were added to the monolayer in a volume of 20 µl at 4° C for 60 min, then washed and solubilized with 0.2% sodium dodecyl sulphate. Radioactivity was measured in a NK 350 scintillation counter. The amount of the specifically-bound LDL was expressed as ¹²⁵I-LDL and ¹²⁵I-acLDL µg/mg protein at a tracer LDL concentration of 50 µg/ml. The proportion of nonspecific binding was 10-15% of the specific binding in the presence of a 15-fold level of non-labelled cold LDL [20]. The specific degradation of native and acLDL was determined at 37° C for 4 h as described above. The level of degraded LDL was determined as the trichloracetic acid-soluble fraction of the supernatant.

Inhibition of cholesterol synthesis

In a volume of 0.6 ml, 50 µg/ml LDL and ¹⁴C-acetate (Amersham) were added to the monocyte suspension (10⁶ cells/0.4 ml). Samples were incubated for 4 h at 37° C. Incubation was stopped and ³H-cholesterol with 3 x 10⁴ cpm activity was added. The samples were then saponified for 90 min at 70° C. The non-saponifiable lipids were extracted in 3 x 2.5 ml n-hexane. The steroid fraction was eluted by acetone diethylether 1:1 on an aluminium oxide column. The radioactivity was measured after drying [21].

Determination of apoE

The amount of apoE was measured using a Hyland PDQ/TM laser nephelometer with a human apoE standard (gift from Prof. H. Kostner, Graz) and anti-human-apoE (sheep) IgG (Immuno AG). The line contained 3.8; 2.85; 1.9; 0.9 and 0.48 µg/ml concentrations of apoE. The MDM monolayers were incubated with 50 µg/ml acLDL in the presence of 5% human serum for 12 h. The removed medium was concentrated on an Amicon 25 filter and the aliquots were used for quantification.

Results

Neither all-trans-RA nor 13-cis-RA preincubation changed ¹²⁵I-LDL and ¹²⁵I-acLDL binding and degradation of MDM₇₂ (Table I). RAs did not influence the cholesterol uptake of these cells either through the specific or the scavenger pathway. LDL incubation decreased the ¹⁴C acetate incorporation of MDM₇₂ from 95.1 ± 9.6 pmol/h/mg protein to 30.2 ± 4.1 pmol/h/mg protein, p < 0.001. RAs changed the LDL-induced inhibition to a negligible extent (all-trans-RA: 25.2 ± 3.4 pmol/h/mg protein, 13-cis-RA: 24.8 ± 3.9 pmol/h/mg protein, p > 0.05). RAs did not have any significant effect on the cholesterol synthesis inhibition caused by native LDL (Fig. 1). Ac-LDL caused 11.2 ± 1.2 µg/12 h/mg of protein apoE secretion. The all-trans-RA incubation decreased this apoE secretion significantly (4.3 ± 0.8 µg/12 h/mg protein, p < 0.05) as did the 13-cis-RA (4.8 ± 0.7 µg/12 h/mg protein, p < 0.05) (Fig. 2).

The results from experiments using monocytes from 5 males suffering from cystic acne treated with Roaccutane^™ are seen in Table II. No significant difference was found in the specific receptor activity (LDL binding and degradation) and the extent of cholesterol synthesis inhibition as compared to the corresponding control, non-treated, age-matched group. There was no alteration in the acLDL binding as regards scavenger receptor activity; acLDL degradation was also decreased only to a slight extent. However, patients 3 and 4 showed considerable differences in the latter. ApoE synthesis decreased significantly in the retinoid treated group.

Discussion

Ligand binding activity of the specific LDL receptor and scavenger LDL receptor and their ligand-incorporating effect (degradation) did not involve RAs in this experiment. RAs did not influence the metabolism of the LDL or acLDL contained in cholesterol, nor did it involve the transport function of the specific and scavenger receptors. There are data in the literature concerning other cell types (e.g. fibroblasts) which show a similar finding even when the LDL particulum itself contains retinoids [22]. Based on these data it can be postulated that hypercholesterolemia during retinoid treatment is not caused by decreased consumption of LDL cholesterol.

RAs were not involved in the inhibitory effect of LDL on endogenous cholesterol synthesis of the macrophages, the biological activity of the specific LDL receptor. In our previous study we found this to be mainly a consquence of intracellular signal transduction [5], as in human platelets [23], but the regulatory effects of endogen sterols on the LDL gene was shown too [24].

Our data from the two types of experiments (exposing the macrophages with RAs in vitro and in vivo) gave similar results in every respect. The only significant change was the decrease in the apoE secretion. The liver is the most important site for apoE production, but macrophages can also synthesize a significant quantity of total apoE [25]. In our opinion, the decrease of monocyte-derived macrophage apoE secretion, caused by LDL acting in a scavenger receptor manner, may serve as a theoretical explanation for the decrease in the HDL cholesterol level observed during retinoid treatment, as less apoE in the periphery makes it less possible for cholesterol to be transported by HDL.

The apoE synthesis can be regulated at several levels (modification of protein configuration, translation and transcription), but this process is not fully understood. The regulating effect of sterols on apoE gene expression [24] and Ap1-like proteins has been described [26] but apart from the transcriptional regulation level, the post-transcriptional control of apoE on THP1 human monocytic cell line is observed during monocyte macrophage differentiation [27] and in other cells [28, 29]. Different cytokines [3, 30, 31], exogenous phospholipase C [32] and platelets [33] can modulate apoE secretion. Protein excretion is also regulated by intracellular signal transduction [32]. Phorbol myristic acetate (PMA) slightly increased the acLDL-induced apoE secretion in our system (data not shown). This effect was opposite to that of RAs. It indicates that RAs may be involved in the signal transduction pathway. Our unpublished data show that RAs inhibited the membrane-bound PKC activity of human monocytes in a 1 µM concentration after 60 min preincubation. The effect of RA on PKC is cell type-specific [34]. Retinoic acid can affect apoE gene transcription through its RAR/RXR nuclear receptor, which belongs to the steroid nuclear hormone receptor superfamily and heterodimerisation of the receptor with other members of the family can occur, as was shown in the case of acyl-CoA oxidase, the rate limiting enzyme of the beta-oxidation pathway [35]. Transcriptional regulation of apoA-I synthesis by retinoid in hepatocytes has been reported [36].

The effect of RAs on apoE secretion has not been published. Data obtained from treated patients also suggest the need for careful evaluation. The point is, to what extent a 3-day culture can be considered as representative of drug treatment. Further studies are needed to determine the mechanism of action of retinoic acids on apoE secretion by macrophages, which may lead to better understanding of the mechanism of retinoid-induced hyperlipidemia in order to produce new retinoid compounds with fewer side effects.

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