Suppression of cholesterol synthesis in cultured fibroblasts from a patient with homozygous familial hypercholesterolemia by her own low density lipoprotein density fraction A possible role of apolipoprotein E

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Biochimicn et Biophysics Acta, 617 (1980) 529-535 o Elsevier/North-Holland Biomedical Press

BBA Report BBA 51273

SUPPRESSION OF CHOLESTEROL SYNTHESIS IN CULTURED FIBROBLASTS FROM A PATIENT WITH HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA BY HER OWN LOW DENSITY LIPOPROTEIN DENSITY FRACTION A POSSIBLE

ROLE OF APOLIPOPROTEIN

E

L. HAVEKES, B.J. VERMEER*, E. DE WIT, J.J. EMEIS, H. VAANDRAGER**, VAN GENT and J.F. KOSTER**

C.M.

Gaubius Institute, Health Research Organization TNO, Herenstraat 5d, 2313 AD Leiden (The Netherlands) (Received September 26th, 1979) Key words: LDL; Hypercholesterolemia; man fibroblast)

Apolipoprotein

E; Cholesterol synthesis; (Hu-

Summary The suppression of cellular cholesterol synthesis by low density lipoprotein (LDL) from a normal and from a homozygous familial hypercholesterolemit subject was measured on normal fibroblasts and on fibroblasts derived from the same homozygous familial hypercholesterolemic patient. On normal fibroblasts both LDL preparations (denisty 1.019 to 1.063 g/ml) exerted a similar suppression of cellular cholesterol synthesis. With the homozygous familial hypercholesterolemic fibroblasts homozygous hypercholesterolemic LDL suppressed the cholesterol synthesis to a much greater extent than did LDL from a normal subject. Analysis of lipid and protein composition of both LDL preparations showed that homozygous hypercholesterolemic LDL differs from normal LDL. In the homozygous hypercholesterolemic LDL preparation the ratio phosphatidylcholine to sphingomyelin is decreased, and even when taking a narrower density range (1.023 to 1.045 g/ml), apolipoprotein E is present. In this homozygous hypercholesterolemic LDL preparation (density range 1.023 to 1.045 g/ml) apolipoprotein E could be present as an integral LDL protein constituent or as an apolipoprotein of an HDL,like lipoprotein class with a floatation density similar to that of LDL. It is *Department of Dermatology. University Medical Centre. Wassenaarseweg 62. 2333 AL Leiden. The Netherlands. **Present address: Department of Biochemistry I. Faculty of Medicine, Erasmus University. P.O. Box 1738.3000 DR Rotterdam, The Netherlands

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suggested that the presence of apolipoprotein E in the LDL density fraction from plasma from this homozygous familial hypercholesterolemic patient could offer an additional means fo: suppression of cellular cholesterol synthesis of this patient. Considerable attention has been paid to the metabolism of low density lipoprotein (LDL) (density range 1.019-1.063 g/ml) by cultured cells from extra-hepatic tissues. In this process LDL binds to the cell surface plasma membrane by a high-affinity interaction and, after its internalization and degradation, suppresses the cellular cholesterol synthesis [ 1] . The high-affinity binding of LDL occurs at receptor sites on the cellular plasma membrane [ 2, 31 by way of its apolipoprotein B moiety [4] . Studies with cultured fibroblasts and lymphocytes from homozygous familial hypercholesterolemic subjects showed that in these subjects high-affinity binding of LDL is deficient [l, 51. High density lipoproteins (HDL) bind to cultured fibroblasts as well, irrespective of whether these cells originate from normal or from homozygous familial hypercholesterolemic subjects [6, 71. The binding of the apolipoprotein E-containing HDL subfraction should be responsible for the observed suppression of the endogenous cholesterol synthesis by HDL [ 81. It is stated that apolipoprotein E is lo- to lOO-fold stronger in binding to the cellular plasma membranes than apolipoprotein B [9] . strain of homozygous familial hypercholesterolemic fibroblasts [lo] . These cells have specifically less binding capacity to normal LDL (7% of control values, measured according to Stein et al. [ 111, at 14 pg protein of ‘*‘I-labeled LDL). Furthermore, the degradation of ‘*%labeled LDL in these cells, measured as trichloroacetic acid soluble radioactivity, was greatly impaired under the conditions applied. We wondered whether the interaction of LDL from our homozygous familial hypercholesterolemic patient (patient LDL) with her own fibroblasts (patient fibroblasts) was greatly impaired as well. Therefore, we measured the suppression of cholesterol synthesis in the fibroblasts from our patient by her own LDL. In control experiments we measured firstly the suppression of the cholesterol synthesis in normal as well as in the patient fibroblasts by LDL isolated from a healthy subject (normal LDL) and secondly the suppression of the cholesterol synthesis in normal fibroblasts by the patient LDL. The results of these experiments are shown in Fig. 1 (for methods and conditions, see legend to Fig. 1). The suppression of the cholesterol synthesis in normal fibroblasts is identical whether normal LDL or patient LDL was used, at least under the conditions applied. However, when these LDL preparations (normal and patient) were tested on the patient fibroblasts a dramatic difference was obtained. On the patient fibroblasts the patient LDL was much more effective than normal LDL in suppressing the cholesterol synthesis (Fig. 1). This difference could be due to differences in apolipoprotein and/or lipid composition of patient LDL compared to normal LDL. For determination of the lipid composition of patient LDL and normal LDL, the respective LDL preparations were isolated by heparin/2 M MgC12 precipitation following ultracentrifugation at density 1.019 g/ml to remove

531

I

1

I

1

20

40

60

80

pg

LDL cholesterol

1

I

100

I

120

ml medium

Fig. 1. The effect of increasing LDL cholesterol concentration on cholesterol synthesis in fibroblasta from a normal subject and from a patient homozygous for familial hypercholesterolemia. Monolayers of fibroblasts were grown to confluence at 3’7’C on pieces of Melinex plastic in 90 mm plastic Petri dishes in 10 ml medium (Ham’s F 10 growth medium containing 15% (v/v) new-born calf serum) in an atmosphere of 95% sir/5% CO,. After confluence, the fibroblasts were incubated for 48 h at 37OC in medium containing 15% (v/v) delipidated new-born calf serum (density fraction > 1.21 g/ml). For the following 24 h the indicated amount of LDL-cholesterol was added. After these 24 h, 1 mM of [14Cl acetate (spec. act. 12 dpm/pmol acetate) was added and the incubation was continued for a further period of 19 h at 37°C. The cholesterol synthesis in this 19 h period was measured as the amount of radioactivity incorporated in cholesterol. Therefore, cells and medium were extracted with a chloroform/methanol mixture and ‘?Slabeled cholesterol was quantitatively determined after isolation by thin layer chromatography (for further details, see Goldstein et al. [121). The cholesterol synthesized in the 19-h period is a measure for the hydroxylmethylglutaryl-CoA reductase activity (rate-limiting enzyme in cholesterol synthesis). After 19 h of incubation, the maximal activity (taken as 100%) was 3.80 and 5.23 pmol/pg cell protein for respectively normal and patient fibroblasta. The LDL preparations for these experiments were obtained by the method of Redgrave 1131 (sliced density range 1.019-1.063 g/ml). These LDL preparations were not contaminated with HDL since apolipoprotein A-l was not present as measured by rocket immunoelectrophoresis (lower limit of sensitivity about 20 I.rg of apolipoprotein A-l per ml). a-, normal fibroblasts with normal LDL or patient LDL; 9-a. patient fibroblasts with patient LDL; and O---O, patient fibroblasts with normal LDL.

and IDL. Lipids were extracted by chloroform/methanol (2:1, v/v). For measuring the phosphatidylcholine: sphingomyelin ratios the lipids were separated by thin layer chromatography as described by Gluck et al. [ 141. The cholesterol: phospholipid ratio is determined by thin layer chromatography as described by Van Gent [ 151. The results of the determination of the lipid compositions of LDL isolated from five normal subject8 and from an obligate heterozygous familial hypercholesterolemic subject (patient’s mother) and from our patient are presented in Table I. The chole8terol:phospholipid ratio and the phosphatidylcholine:sphingomyelin ratio in the patient LDL is significantly different from the respective ratios in normal LDL. These differences in lipid composition between norVLDL

532 TABLE I PLASMA CHOLESTEROL AND WEIGHT RATIOS OF CHOLESTEROL:PHOSPHOLIPID AND PHOSPHATIDYLCHOLINE:SPHINGOMYELIN IN LDL FROM 5 NORMAL SUBJECTS AND FROM A HETEROZYGOUS AND A HOMOZYGOUS FAMILIAL HYPER~HOLESTEROLEMIC PATIENT n = number of subjects. Group

n

Plasma cholesterol (mg/di)

ChoIesterol:phospholipid ratio (w/w)

Phosphatidylchotinexphingomy&in ratio (w/w)

Normal* Heterozygous Homozygous

5 1 1

<250 487 993

1.43 i 0.10 1.26 1.66**

2.16 f 0.42 1.34 1.12**

*Values are mean + s.d. **Significantly different from normal values (P < 0.05).

ma1 and patient LDL preparations are in full agreement with the data of Yadhav and Thompson [ 161. The significant different lipid composition of the patient LDL as compared with normal LDL might be responsible for the much greater effectivity of the patient LDL than normal LDL in suppression of cellular cholesterol synthesis in the patient fibroblasts (Fig. 1). We wondered, if in the LDL preparation from our homozygous familial hypercholesterolemic patient there is an additional apolipoprotein (perhaps apolipoprotein E) which also could be (partly) responsible for an additional suppression of the endogenous cholesterol synthesis in the patient fibroblasts. Therefore, we again isolated LDL from a normal subject, from our homozygous familial hypercholesterolemic patient, and from her mother (an obligate heterozygous familial hypercholesterolemic patient) by sequential ultracentrifugation between the densities 1.023 and 1.045 g/ml. These LDL subfractions were purified from residual plasma proteins by re-centrifugation at the density 1.045 g/ml. This narrower density range was taken to avoid possible contaminations with other classes of lipoproteins. Double immunodiffusion techniques (Ouchterlony) with mono-specific rabbit anti-apolipoprotein 3, anti-apolipoprotein E and anti-apolipoprotein A-l sera in serial dilutions, showed only the presence of apolipoprotein B in all three LDL subfractions, For all three apolipoproteins the lower limits of sensitivity of the double immunodiffusion assay is about 20 to 40 pg of protein per ml. For higher sensitivity, we used an Enzyme-Linked-Immuno-Sorbens-Assay (ELISA) technique as well [ 17,18]. Polystyrene cups (Cooke microtiter) were coated separately with LDL, delipidated VLDL, and delipidated HDL, as a source for apolipoprotein B, apolipoprotein E, and apolipoprotein A-l respectively. In Fig. 2A it is shown that, using LDL (apolipoprotein B) coated cups, the addition of the respective LDL preparations, together with rabbit anti-apolipoprotein B serum, resulted eventually in a decrease in the absorbance at 449 nm. This indicates that, as expected, apolipoprotein B is present in these LDL subfractions. Strikingly, Fig. 2B shows that the patient LDL subfraction as well as the patient’s mother LDL subfraction contain apolipoprotein E, although the latter to a lesser extent. On the contrary, the corresponding normal LDL subfraction doe8 not contain any detectable amount of apolipoprotein E.

533 A

60 -

60

LOL

(mg/ml)

Fig. 2. Determination of apollpoproteins B, E and A-l in normal and patient LDL density fraction by the ELISA technique. Effect of the addition of LDL on the rate of colour development as measured by the absorbance at 449 nm (Alh,1.The LDL preparations (density 1.023 to I.045 g/ml) were isolated by sequential ultracentrifugation. The ELISA technique was carried out as described by Fruchart et al. [17] ; coating was performed with 1 ng protein/ml; S-aminosalic~licacid/H,O, was used as enzyme substrate 1181; the enzyme reaction was allowed to proceed for 16 min at room temperature. (A) LDL coated; anti-apofipoproteln B antiserum. (B) Dalipidated VLDL coated; anti-apollpoprotein E antiserum. (C) Dellpidated HDL coated; anti-apop~poprote~ A-l antiserum. o----O, LDL lsoiated from a normal subject; n---b, LDL isolated from the homozygous familial hypercholesterolemic patient; A--+ LDL isolated from the heterozygous familial hypercholesterofemic patient: a - l, VLDL (in B) or HDL (in Cl added instead of the respective LDL preparations.

The addition of VLDL instead of LDL shows a rapid decrease in the absorbance at 449 nm with increasing the amount of VLDL protein added (Fig. 2B). Based on the protein composition of this VLDL preparation (about 5% (w/w) of which consists of apolipoprotein E as determined by rocket immunoelectrophoresis), it can be calculated that the protein component of the patient LDL and the patients mother LDL consists of about 1 and 0.1% (w/w) of apolipoprotein E, respectively. However, it remained possible that the LDL preparations from these two familial hyper~holesterolemic patients

534

(homozygous and heterozygous) were contaminated with an apolipoprotein E-containing HDL subfraction with low buoyant density like HDLc [ 7,191. If so, these two patient LDL preparations could contain apolipoprotein A-l as well. However, using the sensitive ELISA technique, it is shown that all three LDL preparations contain no detectable amount of apolipoprotein A-l (Fig. 2C). The lower limit of sensitivity of the ELISA assay for apolipoprotein A-l was about 0.6 pg per ml, as shown by the experiment in which HDL was added instead of LDL (Fig. 2C), assuming that about 60% (w/w) of HDL protein consists of apolipoprotein A-l. Although the concentration of apolipoprotein A-l of these patient LDL preparations are below 0.5% (w/w) (calculated from Fig. 2C) it is still possible that these LDL preparations do contain apolipoprotein A-l. Consequently, these LDL preparations (density range 1.023 to 1.045 g/ml) perhaps are contaminated with HDL,, a lipoprotein class which has the same floatation density as LDL and contains mainly apolipoprotein E [ 201. Otherwise it could be possible that these LDL preparations are contaminated with a kind of ‘apo E HDL,‘, a particularly distinctive HDL, which has been isolated from the plasma of dogs fed a coconut-oilcholesterol diet and which contain apolipoprotein E as the only detectable apolipoprotein [ 191. Unfortunately, the concentration of this particular lipoprotein in the patients LDL preparations should have been too low to be detected by immunoelectrophoresis. Taking these results together, there are two possible explanations for the observed difference between normal LDL and LDL isolated from a homozygous familial hypercholesterolemic patient in suppression of the cellular cholesterol synthesis in fibroblasts derived from the same homozygous familial hypercholesterolemic patient (Fig. 1). Firstly, the LDL particles in the patient are different in lipid and apolipoprotein composition, possibly as a result of a decreased fractional catabolic rate of LDL which increases the mean age of the population of LDL particles circulating in plasma [16] . The presence of apolipoprotein E on these LDL particles might be the result of a changed phosphatidylcholine:sphingomyelin ratio. Secondly, the possibility exists that the patient LDL subfraction (density range 1.023 to 1.045 g/ml) is contaminated with an apolipoprotein E-containing lipoprotein class with a floatation density similar to the density of LDL (i.e., HDL or apo E HDL,). Immunoelectronmicroscopic studies done by Vermeer et al. using antiapolipoprotein B antiserum for visualization did not show binding of the patient LDL to her own fibroblast (unpublished results). This negative result support the second possibility. The concentration of apolipoprotein E in the homozygous patient LDL density fraction is about 1% (w/w) (Fig. 2B). Assuming that the ‘apolipoprotein E-mediated’ binding activity to fibroblasts is 10 to 100 times enhanced by comparison with the ‘apolipoprotein B-mediated’ binding of LDL [ 191, the suppression of the intracellular cholesterol synthesis in patient fibroblasts by the patient LDL subfraction (Fig. 1) lies in the expected order of magnitude. We suggest that in our homozygous familial hypercholesterolemic patient the endogenous cholesterol synthesis is (partly) suppressed by means of

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‘apolipoprotein E-mediated’ binding of apolipoprotein E-containing lipoproteins (possibly HDL,-like lipoproteins) having a floatation density equal to that of LDL. Whether this is a general phenomenon in patients with homozygous familial hypercholesterolemia and whether these lipoproteins function similarly in vivo, remains to be investigated with other homozygous familial hypercholesterolemic patients. The authors thank Dr. J.A. Gevers Leuven for providing us with the plasma samples of these familial hypercholesterolemic patient. The Dutch Heart Foundation is acknowledged for partial financial support. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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