Taku Yamamura

Effects of probucol on xanthomata regression in familial hypercholesterolemia

Fifty-one patients with familial hypercholesterolemia were treated for 2 to 4 years with probucol, cholestyramine, clofibrate and compactin in various combinations. Mean baseline serum cholesterol was 359 +/- 10 mg/dl in the heterozygote, and 582 +/- 52 mg/dl in the homozygote patients. We found that a combination of probucol, cholestyramine and compactin decreased serum cholesterol to normal or near normal in most of the heterozygote patients. In 3 severely affected heterozygote and all 8 homozygote patients, adequate cholesterol reduction was only possible with plasmapheresis plus a hypolipidemic agent. Measurement of the Achilles tendon after 12 to 16 months of treatment showed that reductions in thickness occurred in all patients taking probucol, even in a single-drug regimen, in those undergoing plasmapheresis, especially if probucol was used and in those receiving a combination of cholestyramine and compactin. Probucol was most effective in patients who experienced the greatest decreases in high density lipoprotein (HDL) levels, whereas the cholestyramine-compactin combination worked without decreasing HDL concentrations. Combined clofibrate-cholestyramine therapy, by contrast, led to increased tendon thickness in all but 1 patient. It is believed that probucol exerts its positive effect on xanthomata regression by reducing the size of HDL particles, as was shown in this study. It has already been reported that smaller HDL particles are more active in reverse cholesterol transport. The direct peripheral action of probucol may have aided regression as well.

Familial type I hyperlipoproteinemia caused by apolipoprotein C-II deficiency

A study was made on the clinical and biochemical features of siblings of patients with hyperchylomicronemia and its inherited relationship. It was not a case of the classical type of familial LPL deficiency, but of familial apolipoprotein C-II deficiency. The first patient with apolipoprotein C-II deficiency was reported by Breckenridge et al. and our patients provide the basis for the second report of this new disease. Our observations in this study strongly suggest that familial apolipoprotein C-II deficiency is transmitted by an autosomal recessive mode of inheritance and heterozygotes of this disorder have no abnormalities of plasma lipid and lipoproteins in spite of the reduced plasma apolipoprotein C-II.

LDLR Database (second edition): New additions to the database and the software, and results of the first molecular analysis

Mutations in the LDL receptor gene (LDLR) cause familial hypercholesterolemia (FH), a common autosomal dominant disorder. The LDLR database is a computerized tool that has been developed to provide tools to analyse the numerous mutations that have been identified in the LDLR gene. The second version of the LDLR database contains 140 new entries and the software has been modified to accommodate four new routines. The analysis of the updated data (350 mutations) gives the following informations: (i) 63% of the mutations are missense, and only 20% occur in CpG dinucleotides; (ii) although the mutations are widely distributed throughout the gene, there is an excess of mutations in exons 4 and 9, and a deficit in exons 13 and 15; (iii) the analysis of the distribution of mutations located within the ligand-binding domain shows that 74% of the mutations in this domain affect a conserved amino-acid, and that they are mostly confined in the C-terminal region of the repeats. Conversely, the same analysis in the EGF-like domain shows that 64% of the mutations in this domain affect a non-conserved amino-acid, and, that they are mostly confined in the N-terminal half of the repeats. The database is now accessible on the World Wide Web at http://www.umd.necker.fr

A new isoform of apolipoprotein E — Apo E-5 — Associated with hyperlipidemia and atherosclerosis

Heterogeneity of apolipoprotein E (apo E) was analyzed by isoelectric focusing of apo VLDL in patients with hyperlipidemia and/or atherosclerosis. Six major apo E phenotypes were shown, in agreement with the current genetic model which is composed of 3 major apo E isoproteins, apo E-4, apo E-3 and apo E-2, resulting from three apo E alleles, epsilon 4, epsilon 3 and epsilon 2, at a single genetic locus. We recognized an additional apolipoprotein band, which is located basic to apo E-4 on an isoelectric focusing gel, in 3 patients with hyperlipidemia. The new apolipoprotein component, named apo E-5, was identical with ordinary apo E in apparent molecular weight by SDS-polyacrylamide gel electrophoresis and in its interactions with heparin-Sepharose gel and with anti-apo E antibody. This mutant apo E isoprotein had an isoelectric point more basic by one unit of charge than apo E-4. Two of 3 patients had the phenotype E5/3, and the other the phenotype E5/4. Genetic analysis of the apo E phenotypes in family members of the patients indicated the presence of a new apo E allele (epsilon 5) at the same genetic locus as hitherto known alleles. Since most of the subjects above 50 years old with apo E-5 had ischemic heart disease or cerebral infarction, it was suggested that the mutant apo E-5 may possibly be related to the development of atherosclerosis.

Probucol prevents lipid storage in macrophages

Effects of probucol on lipid storage in macrophages in vitro in the presence of acetylated low density lipoprotein (acetyl-LDL) were observed using macrophage-like cells (UE-12) established from a human histiocytic lymphoma cell line (U-937). Under ordinary light microscopy as well as under electron microscopy we found that probucol added to the medium, either in ethanolic solution or bound to LDL, markedly prevented the development of macrophages into foam cells. Microscale enzymatic assay of cholesterol also showed that the intracellular accumulation of esterified cholesterol caused by acetyl-LDL was markedly decreased by the addition of probucol. The concentration of probucol added to the medium was almost comparable to the plasma concentration of the drug usually obtained in patients under treatment with probucol. The possibility that probucol interferes with the binding of acetyl-LDL to the receptors on the cell surface was suggested. The results of the present investigation coincide with the clinical findings that probucol causes a more marked regression of xanthomas than would be expected from the extent of lowering of LDL cholesterol.

Comparison of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins and trioleoylglycerol/phosphatidylcholine-stabilized particles as substrates for lipoprotein lipase

The effect of apolipoproteins C-II and C-III on the lipoprotein lipase-catalyzed hydrolysis of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins and particles of trioleoylglycerol stabilized with a phosphatidylcholine monolayer was investigated. For both triacylglycerol-rich lipoproteins and artificial lipid particles, maximal lipoprotein lipase activity occurred at a constant apolipoprotein C-II/phospholipid mol ratio of 2.0 X 10(-4) and was independent of particle size, indicating that the amount of apolipoprotein C-II bound to the surface of the substrate is important for enzyme activation. The effect of apolipoprotein C-II on lipoprotein lipase activity with apolipoprotein C-II-deficient lipoproteins as substrate was to decrease the apparent Michaelis constant (Kmapp) from 7.1 to 1.0 mM with minor changes on the apparent maximal velocity (Vmax) (22.2 mmol free fatty acid released/h per mg enzyme). In contrast, apolipoprotein C-II increased the apparent Vmax from 2.4 to 20.0 mmol free fatty acid/h per mg enzyme for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol/phospholipid particles with little change in Kmapp (1.0 mM). Addition of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins or high-density lipoproteins to trioleoylglycerol/phospholipid particles in the presence of apolipoprotein C-II inhibited lipoprotein lipase activity. Lipoprotein lipase activity was also inhibited by the addition of a large excess of lipid-free apolipoprotein C-III to the artificial particles. The decrease in lipoprotein lipase activity correlated with the amount of bound apolipoprotein C-II. We suggest that the reported discrepancies on the effect of apolipoproteins C-II and C-III on lipoprotein lipase catalysis is related to differences in substrates and to the amount of added apolipoproteins.

Common Mutations in the Low-Density-Lipoprotein–Receptor Gene Causing Familial Hypercholesterolemia in the Japanese Population

Familial hypercholesterolemia (FH) is a common genetic disorder caused by mutations of the LDL-receptor gene. In the present study, we investigated four Japanese FH homozygotes and identified five point mutations: a splice site mutation in intron 12 (the 1845 + 2 T-->C mutation), a missense mutation in exon 7 (the C317S mutation), a nonsense mutation in exon 17 (the K790X mutation), a missense mutation in exon 14 (the P664L mutation), and a missense mutation in exon 4 (the E119K mutation). We developed simple methods for detecting these mutations. When we examined the presence of these mutations in 24 unrelated FH homozygotes, the 1845 + 2 T-->C mutation was found in 7 of them, and the other four mutations were unique for each proband. We also screened 120 unrelated FH heterozygotes for these mutations and found that the frequencies of the 1845 + 2 T-->C, C317S, K790X, P664L, and E119K mutations were 13.3% (16/120), 6.7% (8/120), 6.7% (8/120), 3.3% (4/120), and 1.7% (2/120), respectively. These mutations were found in more than 30% of unrelated Japanese FH patients. By using the detection methods developed in this study, the diagnosis of more than 30% of the genetic bases of Japanese FH heterozygotes is expected.

Acquisition of secretion of transforming growth factor-beta 1 leads to autonomous suppression of scavenger receptor activity in a monocyte-macrophage cell line, THP-1.

Macrophage cells derived from the human monocytic leukemia cell line, THP-1, accumulate esterified cholesterol when cultivated in the presence of acetylated low density lipoprotein (Ac-LDL) through scavenger receptors (ScR). In the present study, we isolated a subtype of THP-1 cells that failed to accumulate esterified cholesterol when cultivated in the presence of Ac-LDL. The cells had negligible amounts of cell association and degradation of Ac-LDL compared with the parent THP-1 cells. The subtype THP-1 cells did not express ScR mRNA as well as that of lipoprotein lipase. In contrast, the expression of apolipoprotein E mRNA was greater than that found in parent THP-1 cells. The culture medium of subtype THP-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate inhibited the uptake of Ac-LDL and the expression of ScR in parent THP-1 cells. After a 48-h incubation in the culture medium containing 12-O-tetradecanoylphorbol-13-acetate, the culture medium of differentiated subtype THP-1 cells contained 6.9 ng/ml transforming growth factor (TGF)-β1, while that of parent THP-1 cells secreted below detection level, which was less than 3 ng/ml. This inhibitory effect of the conditioned medium on the expression of ScR in parent THP-1 cells was abolished by pretreatment of the culture medium with anti-TGF-β1 antibodies. Parent THP-1 cells expressed as much TGF-β1 mRNA as sTHP-1 cells after stimulation of differentiation. Although the precursor forms of TGF-β1 that were synthesized in both parent and subtype THP-1 cells were of similar size and were expressed at similar levels, latent TGF-β1-binding protein, which is necessary for the secretion of TGF-β1, could only be co-immunoprecipitated with anti-TGF-β1 antibody from subtype THP-1 cells. This suggests that subtype THP-1 cells secrete TGF-β1 into the medium by forming a functional complex with the latent TGF-β1-binding protein. We conclude that subtype THP-1 cells could not take up Ac-LDL because ScR was inhibited (leading to a loss of function) caused by the secreted TGF-β1.

Low density lipoprotein (LDL) binding affinity for the LDL receptor in hyperlipoproteinemia

We measured the binding affinity of low density lipoprotein (LDL) for the LDL receptor in patients with various types of hyperlipoproteinemia and investigated the effects of LDL lipid composition and particle size on receptor affinity. LDL (1.019 < d < 1.063) was isolated by sequential ultracentrifugation from the serum of normolipidemic controls and patients with hyperlipoproteinemia. Patients with type IIa hyperlipoproteinemia had LDL with a similar receptor affinity to that of normal LDL. However, patients with hypertriglyceridemia (type IIb and type IV hyperlipoproteinemia) had LDL with a low receptor affinity, and the degree of the reduction in affinity paralleled the severity of the hypertriglyceridemia. The LDL of hypertriglyceridemic patients was rich in protein and triglycerides, had a low content of cholesterol and phospholipids, and was smaller than normal, thus resembling the atherogenic lipoprotein known as small, dense LDL. These abnormalities were observed even in patients with mild hypertriglyceridemia regardless of their serum cholesterol levels. The degree of alteration in LDL lipid composition and particle size was strongly associated with the reduction of LDL receptor affinity. We also examined the effects of two lipid-lowering agents (bezafibrate and probucol) on the characteristics of LDL. LDL receptor affinity was only improved when the lipid composition and particle size were normalized by drug therapy. Although it has been reported that decreased cholesteryl ester transfer protein (CETP) activity results in the formation of small LDL, plasma CETP activity was normal in the hyperlipoproteinemic patients and the normalization of LDL characteristics by drug therapy was not accompanied by an increase of CETP activity. Our results suggested that an abnormal lipid composition and/or small particle size might cause a decrease in the receptor affinity of LDL. These structural and functional abnormalities were reversed by drug therapy, underlining the importance of treating hypertriglyceridemia for the prevention of atherosclerosis.

Enhanced binding activity of an apolipoprotein E mutant, APO E5, to LDL receptors on human fibroblasts.

We have previously demonstrated an apolipoprotein E (apo E) mutant, apo E5, associated with hyperlipidemia and atherosclerotic vascular diseases. To investigate the possible mechanism of apo E5 involvement in the development of hyperlipidemia, we have isolated the apo E isoprotein and determined its binding activity to LDL (low density lipoprotein) receptors. It was shown that the apo E5 isoprotein was two times more active for binding to LDL receptors than apo E3. The concentration of apo E, at which 50% of 125I-labeled LDL bound to the receptor was displaced, was 29 ng/ml for apo E5 and 63 ng/ml for apo E3. This result suggests that the high affinity of apo E5 to LDL receptors results in a high uptake of apo E5-containing lipoproteins by the liver and leads to a down-regulation of LDL receptors in the liver. We can postulate that individuals with apo E5 are susceptible to hypercholesterolemia and in consequence to atherosclerosis.