Ryoyu Takeda

Molecular basis of lipid transfer protein deficiency in a family with increased high-density lipoproteins.

PLASMA high density lipoproteins (HDL) are a negative risk factor for atherosclerosis. Increased HDL is sometimes clustered in families, but a genetic basis has never been clearly documented1. The plasma cholesteryl ester transfer protein (CETP) catalyses the transfer of cholesteryl ester from HDL to other lipoproteins and therefore might influence HDL levels2. Using monoclonal antibodies, we show that CETP is absent in two Japanese siblings who have markedly increased and enlarged HDL. Furthermore, they are homozygous for a point mutation in the 5′-splice donor site of intron 14 of the gene for CETP, a change that is incompatible with normal splicing of pre-messenger RNA3. The results indicate that the family has an inherited deficiency of CETP due to a gene splicing defect, and illustrate the key role that CETP has in human HDL metabolism.

Intermediate-density lipoprotein and cholesterol-rich very low density lipoprotein in angiographically determined coronary artery disease.

The relationship between the concentrations of intermediate-density lipoprotein (IDL) and other lipoproteins and the extent of coronary artery disease (CAD) was studied in 182 consecutive patients evaluated by selective coronary cineangiography. On univariate analysis, the extent of CAD correlated significantly and positively with very low density lipoprotein (VLDL) cholesterol, IDL cholesterol and lowdensity lipoprotein (LDL) cholesterol, and negatively with high-density lipoprotein (HDL) cholesterol. Analysis of four subgroups divided by IDL cholesterol and LDL cholesterol levels indicated that moderately increased levels of IDL cholesterol were closely associated with a high frequency of CAD. Moreover, multivariate regression analysis demonstrated that IDL cholesterol for men, LDL cholesterol for men and women and HDL cholesterol for men were significant variables of use in the final weighting procedure. IDL cholesterol was closely associated with cholesterol-rich VLDL. This study shows that IDL and cholesterol-rich VLDL combine to contribute to the development of CAD.

Genetic cholesteryl ester transfer protein deficiency caused by two prevalent mutations as a major determinant of increased levels of high density lipoprotein cholesterol.

Genetic determinants of HDL cholesterol (HDL-C) levels in the general population are poorly understood. We previously described plasma cholesteryl ester transfer protein (CETP) deficiency due to an intron 14 G(+1)-to-A mutation(Int14 A) in several families with very high HDL-C levels in Japan. Subjects with HDL-C > or = 100 mg/dl (n = 130) were screened by PCR single strand conformational polymorphism analysis of the CETP gene. Two other mutations were identified by DNA sequencing or primer-mediated restriction map modification of PCR products: a novel intron 14 splice donor site mutation caused by a T insertion at position +3 from the exon14/intron14 boundary (Int14 T) and a missense mutation (Asp442 to Gly) within exon 15 (D442G). The Int14 T mutation was only found in one family. However, the D442G and Int14 A mutations were highly prevalent in subjects with HDL-C > or = 60 mg/dl, with combined allele frequencies of 9%, 12%, 21% and 43% for HDL-C 60-79, 80-99, 100-119, and > or = 120 mg/dl, respectively. Furthermore, prevalences of the D442G and Int14 A mutations were extremely high in a general sample of Japanese men (n = 236), with heterozygote frequencies of 7% and 2%, respectively. These two mutations accounted for about 10% of the total variance of HDL-C in this population. The phenotype in a genetic compound heterozygote (Int14 T and Int14 A) was similar to that of Int14 A homozygotes (no detectable CETP and markedly increased HDL-C), indicating that the Int14 T produces a null allele. In four D442G homozygotes, mean HDL-C levels (86 +/- 26 mg/dl) were lower than in Int14 A homozygotes (158 +/- 35 mg/dl), reflecting residual CETP activity in plasma. In 47 D442G heterozygotes, mean HDL-C levels were 91 +/- 23 mg/dl, similar to the level in D442G homozygotes, and significantly greater than mean HDL-C levels in Int14 A heterozygotes (69 +/- 15 mg/dl). Thus, the D442G mutation acts differently to the null mutations with weaker effects on HDL in the homozygous state and stronger effects in the heterozygotes, suggesting dominant expression of a partially defective allele. CETP deficiency, reflecting two prevalent mutations (D442G and Int14 A), is the first example of a genetic deficiency state which is sufficiently common to explain a significant fraction of the variation in HDL-C in the general population.

Deficiency of Serum Cholesteryl-Ester Transfer Activity in Patients with Familial Hyperalphalipoproteinaemia

Lipoprotein patterns and cholesteryl ester transfer activity (CETA) were examined in 2 patients with familial hyperalphalipoproteinaemia (FHALP). The proband was a healthy 58-year-old Japanese male who had an HDL cholesterol of 7.83 mmol/l (301 mg/dl). His sisters HDL cholesterol was 4.52 mmol/l (174 mg/dl), which suggested that both were homozygous carriers of FHALP. In both subjects HDL showed a high cholesterol/apo A-I ratio and appeared to be a larger-sized particle than normal HDL on agarose gel chromatography. Two of the probands children showed higher HDL cholesterol levels (1.74 mmol/l, 2.16 mmol/l) than normal, but another 2 children showed normal levels (1.48 mmol/l, 1.40 mmol/l). However, the ratios of HDL cholesterol to total cholesterol and to apo A-I in all children were higher than normal. These data suggest, but do not prove, that all his children were heterozygotes. Apo B levels in all of the family members studied were lower than normal (47-80 mg/dl). Deceased members of the same family had not died from cardiovascular disease. Cholesteryl-ester transfer activity was studied in both patients. When serum or lipoprotein deficient serum (d greater than 1.21) and [3H]cholesteryl ester labelled HDL3 were incubated in the presence of an LCAT inhibitor, there was no evidence of cholesteryl ester transfer from HDL to VLDL and/or LDL, unlike normal subjects. The deficiency of CETA in these patients with FHALP presumably accounted for the increase in particle size and cholesterol enrichment of HDL.

Effects of an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme a reductase on serum lipoproteins and ubiquinone-10 levels in patients with familial hypercholesterolemia

: We studied the effects of ML-236B, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, on serum levels of lipoproteins and ubiquinone-10-in seven heterozygous patients with familial hypercholesterolemia. ML-236B was given at doses of 30 to 60 mg per day for 24 weeks. Serum cholesterol decreased from 390 +/- 9 to 303 +/- 8 mg per deciliter (101 +/- 0.2 to 7.88 +/- 0.2 mmol per liter, mean +/- S.E.M.; p less than 0.001) and serum triglyceride decreased from 137 +/- 18 to 87 +/- 9 mg per deciliter (1.55 +/- 0.20 to 0.98 +/- 0.01 mmol per liter; p less than 0.05). Intermediate-density-lipoprotein (DL) cholesterol, IDL triglyceride, low-density-lipoprotein (LDL) cholesterol, and LDL triglyceride decreased significantly (p less than 0.01, P less than 0.001, and P less than 0.001, respectively). However, there were no significant changes in very-low-density-lipoprotein (VLDL) cholesterol and triglyceride or high-density-lipoprotein (HDL) cholesterol. Serum ubiquinone-10 levels did not change, and LDL levels of ubiquinone-10 decreased by 50 per cent, from 0.39 +/- 0.07 to 0.20 +/- 0.01 microgram per milliliter (P less than 0.05). No adverse effects were observed. We conclude that ML-236B is effective in lowering serum cholesterol without lowering serum ubiquinone-10 in heterozygous patients with familial hypercholesterolemia.

Reduction of serum cholesterol in heterozygous patients with familial hypercholesterolemia. Additive effects of compactin and cholestyramine

We studied the effects of the bile acid sequestrant cholestyramine, alone and in combination with the experimental agent compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on serum levels of lipoproteins in 10 heterozygous patients with familial hypercholesterolemia. After cholestyramine treatment alone for 2 to 16 months, serum total and low-density lipoprotein cholesterol decreased by 20 and 28 per cent, respectively. With the addition of compactin for 12 weeks there was a 39 per cent total decrease in serum cholesterol from the control value--from 356 +/- 14 to 217 +/- 10 mg per deciliter (9.27 +/- 0.36 to 5.64 +/- 0.26 mmol per liter [mean +/- S.E.M.]; P less than 0.001)--and a 53 per cent decrease in low-density lipoprotein cholesterol--from 263 +/- 13 to 125 +/- 10 mg per deciliter (6.84 +/- 0.34 to 3.25 +/- 0.26 mmol per liter; P less than 0.001). High-density lipoprotein cholesterol, which had increased during cholestyramine treatment, remained at its higher level. No adverse effects were observed. If long-term safety can be demonstrated, the compactin-cholestyramine regimen may prove useful in heterozygous familial hypercholesterolemia. prove useful in heterozygous familial hypercholesterolemia.

Development of coronary heart disease in familial hypercholesterolemia.

We studied the development of coronary artery disease in 10 homozygous and 692 heterozygous patients with familial hypercholesterolemia. Seventy-five (22%) male heterozygotes and 35 (10%) female heterozygotes were affected by myocardial infarction, which was first noted in men in the 3rd decade of life and in women in the 4th decade of life. Thirty-eight (70%) out of the deceased 54 heterozygous patients died of coronary heart disease. The mean age at death was significantly less in male heterozygotes (54 years) than in female heterozygotes (69 years). Five homozygous and 105 male and 56 female heterozygous patients received coronary angiographic evaluation. The regression equations between age (X) and coronary stenosis index (Y) obtained by assigning score (0 to 5) to each of 15 coronary artery segments were Y = 1.57X - 20.43 (r = 0.956, p less than 0.05) in the homozygotes, Y = 0.52X - 9.11 (r = 0.438, p less than 0.001) in the male heterozygotes, and Y = 0.47X - 12.54 (r = 0.343, p less than 0.01) in the female heterozygotes. From these data, we can assume that coronary artery stenosis detectable by angiography will occur after 17 and 25 years of age in male and female heterozygotes, respectively, and the treatment of heterozygotes with lipid-lowering drugs can be delayed until late adolescence.

A new low density lipoprotein apheresis system using two dextran sulfate cellulose columns in an automated column regenerating unit (LDL continuous apheresis)

We describe a new low density lipoprotein (LDL) apheresis system using dextran sulfate cellulose column in an automated column regenerating unit (LDL continuous apheresis). Two columns containing 150 ml of dextran sulfate cellulose were used, and the whole extracorporeal circulation was about 400 ml in volume. After 600 ml of plasma was adsorbed into the first column, the second column was used as an adsorbent and meanwhile the first column was regenerated. Thus, the 2 columns were used alternately without losing the potency of the columns. As the apparatus was automatically controlled by a computerized unit, no extra manipulation is necessary compared with the conventional single-column method. By treating 4-5 liters of plasma, non-high density lipoprotein (HDL)-cholesterol levels decreased by 63-71%, and HDL-cholesterol levels remained unchanged. Thus, this new method of LDL apheresis can safely reduce LDL-cholesterol to any desired level and will be applicable for the treatment of child and adult family hypercholesterolemic patients with severe coronary heart disease.

Vascular Aldosterone in Genetically Hypertensive Rats

We have reported that aldosterone is synthesized and cytochrome P450aldo mRNA exists in the vasculature. To clarify the pathophysiological role of vascular aldosterone in hypertension, we compared aldosterone production in the mesenteric arteries of stroke-prone spontaneously hypertensive rats (SHRSP) with that in Wistar-Kyoto rats (WKY). The expressions of mRNA of cytochrome P450aldo, mineralocorticoid receptor, and alpha 1, Na,K-ATPase in the mesenteric arteries were compared between the two groups. Aldosterone concentration in the perfusate of the vasculature was measured by radioimmunoassay after purification with high-performance liquid chromatography. Cytochrome P450aldo and mineralocorticoid receptor mRNA levels were quantified by Southern blot analysis of the products of reverse-transcribed polymerase chain reaction. Levels of alpha 1 Na,K-ATPase mRNA were measured by Northern blot analysis. Vascular aldosterone and cytochrome P450aldo mRNA levels of 2-week-old SHRSP were significantly increased compared with those of age-matched WKY. However, vascular aldosterone in 4- and 9-week-old SHRSP did not differ from that in age-matched WKY. Expression levels of mineralocorticoid receptor mRNA in the vasculature of 4- and 9-week-old SHRSP were significantly increased compared with those in age-matched WKY. Concentrations of vascular alpha 1 Na,K-ATPase mRNA of 2-, 4-, and 9-week-old SHRSP also were significantly higher than those in age-matched WKY. These results suggest that vascular aldosterone contributes to the pathophysiology of hypertension in SHRSP in the early stage.

Production of endothelin-1 from the mesenteric arteries of streptozotocin-induced diabetic rats

Release of endothelin-1 (ET-1) from the mesenteric arteries of Wistar rats with streptozotocin-induced diabetes (STZ-DM) rats and nondiabetic rats was measured by a specific enzyme immunoassay following purification using an immunoaffinity column. The mesenteric arteries from STZ-DM rats released a significantly higher amount of ET-1 as compared to control rats (35.8 +/- 2.8 vs 14.9 +/- 2.0 pg/1hr, p less than 0.05). The plasma level of ET-1 in STZ-DM rats was also elevated to a significant extent as compared to controls (5.1 +/- 0.4 vs 3.0 +/- 0.4 pg/ml, p less than 0.05). The systolic blood pressure of STZ-DM rats was significantly higher than of the controls (p less than 0.05). The increased level of plasma ET-1 as well as its release from the mesenteric artery of STZ-DM rats may suggest its release following damage to the endothelium caused by diabetes and/or by associated changes in blood pressure.