Toshinori Higashikata

Circulating matrix metalloproteinases and their inhibitors in premature coronary atherosclerosis

Abstract To investigate the clinical significance of circulating matrix metalloproteinases (MMPs) and their tissue inhibitos (TIMPs) in patients with premature coronary atheroscrelosis, we studied 53 consecutive male patients with angiographically defined premature (<65 years) and stable coronary artery disease. Plasma levels of MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 were determined in peripheral blood by a sandwich enzyme immunoassay, and the results were compared with those from 133 age-matched control males. There were significant differences in all the MMPs and TIMPs (p<0.001) between patients and controls. In the patient group, the levels of MMP-9 (mean±SD (ng/ml) 27.2±15.2/21.8±15.2) and TIMP-1 (130.4±55.7/94.5±26.3) were significantly higher, and the levels of MMP-2 (632.5±191.6/727.6±171.4), MMP-3 (53.1±31.2/79.6± 29.9), and TIMP-2 (24.7±15.2/35.4±16.4) were significantly lower than those of controls. We found significant positive correlation between plasma MMP-9 levels and low-density lipoprotein (LDL)-cholesterol levels (Rs=0.168, p=0.022), and significant negative correlation between plasma MMP-9 levels and high-density lipoprotein (HDL)-cholesterol levels (Rs=−0.164, p=0.026) by Spearman rank correlation test. In contrast, plasma MMP-2 (Rs=0.181, p=0.014) and MMP-3 (Rs=0.260, p=0.0004) levels were positively correlated with HDL-cholesterol levels. TIMP-2 levels were negatively correlated with total cholesterol (Rs=−0.197, p=0.007) and LDL-cholesterol (Rs=−0.168, p=0.022) levels. These results suggest that the circulating levels of MMPs and TIMPs are altered in patients with premature coronary atherosclerosis and that plasma lipoprotein cholesterol levels correlate with these, possibly as a result of the lipoprotein-vessel wall interactions.

Association of Estrogen Receptor-α Gene Polymorphisms With Coronary Artery Disease in Patients With Familial Hypercholesterolemia

To investigate the association of estrogen receptor (ER)-α gene polymorphisms with coronary artery disease (CAD), we studied 197 men and 98 postmenopausal women with heterozygous familial hypercholesterolemia. We examined the known polymorphisms, including Pvu II, Xba I, TA repeat, and CA repeat, and identified 6 novel polymorphisms in the ER-α gene. The distributions of −1989T/G (a novel polymorphism in promoter B) and Xba I in intron 1 were associated with CAD in postmenopausal women and in men, with a higher frequency of the G/G genotype ( P =0.03) or X1/X1 genotype ( P =0.02) in the CAD group. The frequency of alleles of TA repeats >17 was found to be significantly higher in postmenopausal women with CAD than in those without CAD ( P =0.04), but not in men. Logistic regression analysis with all coronary risk factors as covariates showed that the G/G genotype was a higher risk for CAD (odds ratio 4.5, 95% CI 1.0 to 19.5; P =0.04) but that X1/X1 was not. We conclude that −1989T/G or its linked polymorphisms in the ER-α gene may confer risk for CAD and that the G/G genotype may be an independent predictor for CAD in patients with familial hypercholesterolemia.

Long-term treatment with pitavastatin (NK-104), a new HMG-CoA reductase inhibitor, of patients with heterozygous familial hypercholesterolemia.

The clinical efficacy and safety of pitavastatin (NK-104), a novel HMG-CoA reductase inhibitor, during long-term treatment, were examined in 25 patients (male/female=11/14, mean age=53+/-13 (mean+/-SD) years) with heterozygous familial hypercholesterolemia (FH). After a period on placebo of >4 weeks, 2 mg/day of pitavastatin was administered for 8 weeks, and the dose was increased to 4 mg/day for up to 104 weeks. Total cholesterol (TC) decreased by 31% from the initial value of 340+/-57 to 237+/-40 mg/dl (P<0.0001) at week 8. During treatment with the higher dose, TC decreased even further to 212+/-35 mg/dl at week 12; it decreased by 37% from the initial value (P<0.0001). Similarly, the baseline low-density lipoprotein (LDL)-cholesterol (LDL-C) decreased by 41% at week 8, and by 49% at week 12, from 267+/-61 mg/dl at baseline. These findings indicate a dose-dependent effect of the drug on TC and LDL-C concentrations. To examine whether the levels of circulating matrix metalloproteinases (MMPs) and their endogenous inhibitors (tissue inhibitors of metalloproteinases: TIMPs) are altered during lipid-lowering therapy, we also measured their plasma levels. The mean levels of MMP-2 and -3 were significantly increased. No significant alteration was found in MMP-9, TIMP-1 and -2 levels. As for the safety of pitavastatin, adverse reactions were observed in one case (4%) of subjective and objective symptoms. The effects of pitavastatin on TC and LDL-C were stable during long treatment of patients with heterozygous FH.

Molecular genetic analysis of familial hypercholesterolemia: spectrum and regional difference of LDL receptor gene mutations in Japanese population

To determine the molecular basis of familial hypercholesterolemia (FH) in Japan, 200 unrelated patients with clinically diagnosed heterozygous FH were screened for mutations in coding and promoter region of the low density lipoprotein (LDL) receptor gene using denaturing gradient-gel electrophoresis (DGGE), DNA sequencing and Southern blotting analysis. About 37 different mutations in the LDL receptor gene were identified in 125 (62.5%) of the patients, 22 of these mutations have not been described before. The most common mutations were K790X (19.5%), P664L (6.0%), FH-Tonami-1 (6.0%), IVS15-3C>A (5.5%) and FH-Tonami-2 (4.5%), whereas the other mutations were rare. No apolipoprotein B (apoB) mutations responsible for familial ligand-defective apoB-100 (FDB) were identified. Polymorphisms of apolipoprotein E (apoE) and scavenger receptor class B type I (SR-BI) were observed to have minor effects on the lipid and lipoprotein profile. In 75 (32.5%) of the FH patients, LDL receptor gene mutations could not be identified. These patients had significantly lower total cholesterol (7.71+/-1.64 vs. 8.68+/-1.47 mmol/l, P<0.001) and LDL-cholesterol (6.02+/-1.51 vs. 6.87+/-1.47 mmol/l, P<0.001) in plasma, also a lower incidence of coronary heart disease (CHD) (22 vs. 29%, P=0.05) compared with patients with a LDL receptor gene mutation, suggesting that besides LDL receptor, defect of other genes involved in LDL metabolism may be a cause of FH with a milder phenotypic expression in Japanese population.

ATP-binding cassette transporter G8 M429V polymorphism as a novel genetic marker of higher cholesterol absorption in hypercholesterolaemic Japanese subjects.

The ratio of serum plant sterols to cholesterol is positively correlated with the fractional cholesterol absorption, whereas serum precursors of cholesterol synthesis are positively correlated with cholesterol synthesis. Recently, two ABC (ATP-binding cassette) transporters, ABCG5 and ABCG8, have been described as playing an important role in the absorption and excretion of sterols. In the present study, we tested the hypothesis that genetic variation in ABCG5/ABCG8 influences the levels of serum plant sterol (sitosterol) and cholesterol precursor (lathosterol) in Japanese primary hypercholesterolaemic patients (n = 100). We identified a novel mutation [859T/C (C287R)] and a novel polymorphism [1285A/G (M429V)] at the ABCG5/ABCG8 loci, as well as four polymorphisms reported previously [1810C/G (Q604E), 161G/A (C54Y), 1199C/A (T400K) and 1895C/T (A632V)]. In carriers of the novel M429V variant, the serum level of sitosterol and the sitosterol/cholesterol ratio were significantly higher than those in non-carriers (3.64 compared with 2.56 microg/ml, and 1.45 microg/mg compared with 1.00 microg/mg respectively; P < 0.01 for both), and serum lathosterol tended to be lower (1.95 microg/ml compared with 3.03 microg/ml; P = 0.08), whereas no significant difference was observed in other lipid profiles. These four polymorphisms (1810C/G, 161G/A, 1199C/A and 1285A/G) generated six haplotypes, and the C/G/C/G haplotype was significantly associated with a higher sitosterol level and sitosterol/cholesterol ratio compared with the other five haplotypes (P < 0.05 for both). We conclude that, in 8% of patients with hypercholesterolaemia, the novel ABCG8 M429V variant was associated with higher cholesterol absorption efficiency. Future studies should investigate whether these findings have implications for the optimal cholesterol-lowering drug treatment in hypercholesterolaemic patients.

Efficacy and Safety of Alirocumab in Japanese Patients With Heterozygous Familial Hypercholesterolemia or at High Cardiovascular Risk With Hypercholesterolemia Not Adequately Controlled With Statins – ODYSSEY JAPAN Randomized Controlled Trial –

BACKGROUND The ODYSSEY Japan study was designed to demonstrate the reduction in low-density lipoprotein cholesterol (LDL-C) by alirocumab as add-on to existing lipid-lowering therapy in Japanese patients with heterozygous familial hypercholesterolemia (heFH) or non-FH at high cardiovascular risk who require additional pharmacological management to achieve their LDL-C treatment goal (<2.6 or <3.1 mmol/L, depending on risk category). METHODSANDRESULTS This randomized, double-blind, parallel-group, 52-week study was conducted in Japan. Patients (n=216) with heFH, non-FH at high cardiovascular risk with coronary disease, or classified as category III were enrolled. The prespecified safety analysis was done after the last patient completed 52 weeks. Patients were randomized (2:1, alirocumab:placebo) with stratification for heFH to s.c. alirocumab (75 mg every 2 weeks [Q2 W] with increase to 150 mg if week 8 LDL-C ≥2.6/3.1 mmol/L) or placebo for 52 weeks plus stable statin therapy. At week 24, mean±SE change in LDL-C from baseline was -62.5±1.3% in the alirocumab group and 1.6±1.8% in the placebo group (difference, -64.1±2.2%; P<0.0001); the reduction was sustained to week 52 (alirocumab, -62.5±1.4%; placebo, -3.6±1.9%). No patterns were evident between treatment groups for adverse events at 52 weeks. CONCLUSIONS In high-risk Japanese patients with hypercholesterolemia on stable statin therapy, alirocumab markedly reduced LDL-C vs. placebo and was well tolerated over 52 weeks. (Circ J 2016; 80: 1980-1987).

Low-density lipoprotein receptor genotype-dependent response to cholesterol lowering by combined pravastatin and cholestyramine in familial hypercholesterolemia

We compared the effects of cholesterol-lowering therapy on 2 patient groups genetically defined as heterozygous for familial hypercholesterolemia (FH), 5 with a deletion of exon 15 (FH(Tonami-1)), and 7 with a point mutation at codon 664 (FH(Kanazawa-2)). There were significant differences in both serum and low-density lipoprotein cholesterol reductions between the 2 groups after combination therapy with pravastatin and cholestyramine, and the overall effect of genotype on serial changes in both was significant.

Cholesterol efflux from J774 macrophages and Fu5AH hepatoma cells to serum is preserved in CETP-deficient patients

BACKGROUND The role of CETP in the development of atherosclerosis is debatable, and few data exist regarding the total impact of CETP inhibition on cholesterol efflux. METHODS Acceptor capacities of whole serum and HDL subfractions separated by HPLC were compared using 2 different cell systems. Subjects with CETP deficiency (2 homozygous, 1 compound heterozygous, and 5 heterozygous) were analyzed along with 10 normolipidemic controls. The fractional efflux from cholesterol-labeled Fu5AH hepatoma cells was determined to be SR-BI mediated. The efflux difference between control and liver X receptor (LXR) agonist-induced ABCA1-upregulated J774 macrophages was considered as a measure of ABCA1-mediated efflux. RESULTS For the Fu5AH cell system, the total acceptor capacities of whole serum and HPLC-separated HDL fraction 2 obtained from the homozygous subjects were 38% and 116% higher than the corresponding values for the controls, respectively (p<0.05). For the J774 cell system, the total acceptor capacities of whole serum and HPLC-separated HDL fractions were similar among the CETP-deficient subjects and controls. CONCLUSIONS Serum from homozygous subjects with CETP-null defects exhibited enhanced acceptor capacity via an SR-BI dependent pathway, which is regulated by the middle HPLC-separated HDL fraction. Further, the cholesterol acceptor capacity of serum obtained from patients having complete and partial CETP deficiency was preserved via an ABCA1-dependent pathway.

Double deletions and missense mutations in the first nucleotide-binding fold of the ATP-binding cassette transporter A1 (ABCA1) gene in Japanese patients with Tangier disease

AbstractTangier disease (TD) is a rare autosomal recessive disease characterized by plasma high-density lipoprotein deficiency caused by an ATP-binding cassette transporter A1 (ABCA1) gene mutation. We describe three different mutations in Japanese patients with TD. The first patient was homozygous for double deletions of 1221bp between intron 12 and 14 and 19.9kb between intron 16 and 31. The breakpoint sequence analyses suggest that it is a simultaneous event caused by double-loop formation through multiple Alu. The second patient was homozygous for a novel mutation of A3198C in exon 19, resulting in Asn935His. The third patient was homozygous for A3199G of exon 19 that leads to Asn935Ser, which is the same mutation found in German and Spanish families. Both Asn mutations involved Walker A motif of the first nucleotide-binding fold.

CETP (cholesteryl ester transfer protein) promoter −1337 C>T polymorphism protects against coronary atherosclerosis in Japanese patients with heterozygous familial hypercholesterolaemia

CETP (cholesteryl ester transfer protein) and HL (hepatic lipase) play a role in the metabolism of plasma lipoproteins, but the effects of CETP and LIPC (gene encoding HL) genotypes on coronary atherosclerosis may be dependent on LDL (low-density lipoprotein)-receptor activity. Recently, the -1337 C>T polymorphism in the CETP gene has been reported in REGRESS (Regression Growth Evaluation Statin Study) to be a major determinant of promoter activity and plasma CETP concentration. In the present study, we have investigated the effects of the CETP promoter -1337 C>T and LIPC promoter -514 C>T polymorphisms on serum lipid profiles and risk of coronary atherosclerosis in 206 patients (154 males) with heterozygous FH (familial hypercholesterolaemia). To evaluate coronary atherosclerosis, we used CSI (coronary stenosis index) calculated from coronary angiograms. The CETP -1337 T allele was less frequent in subjects with a CSI > or =14 (mean value) in the group with coronary artery disease (P=0.04, as determined by chi(2) test). ANOVA revealed that HDL-C (high-density lipoprotein-cholesterol) and triacylglycerol (triglyceride) levels were not significantly higher in the presence of the CETP promoter -1337 T allele. Combined with LIPC promoter polymorphisms, HDL-C levels were highest and CSI were lowest with CETP -1337 CT+TT and LIPC -514 CC genotypes, but a significant interaction was not shown. A multiple logistic regression analysis revealed that, in patients with coronary atherosclerosis, the CETP- 1337 CC genotype was a significant genetic risk factor in FH (odds ratio=2.022; P=0.0256). These results indicate that the CETP promoter -1337C>T polymorphism is associated with the progression of coronary atherosclerosis in Japanese patients with FH, independent of HDL-C and triacylglycerol levels.