Hiroaki Yagyu

Embryonic Lethality and Defective Neural Tube Closure in Mice Lacking Squalene Synthase

Squalene synthase (SS) catalyzes the reductive head-to-head condensation of two molecules of farnesyl diphosphate to form squalene, the first specific intermediate in the cholesterol biosynthetic pathway. We used gene targeting to knock out the mouse SS gene. The mice heterozygous for the mutation (SS+/−) were apparently normal. SS+/− mice showed 60% reduction in the hepatic mRNA levels of SS compared with SS+/+ mice. Consistently, the SS enzymatic activities were reduced by 50% in the liver and testis. Nevertheless, the hepatic cholesterol synthesis was not different between SS+/− and SS+/+ mice, and plasma lipoprotein profiles were not different irrespective of the presence of the low density lipoprotein receptor, indicating that SS is not a rate-limiting enzyme in the cholesterol biosynthetic pathway. The mice homozygous for the disrupted SS gene (SS−/−) were embryonic lethal around midgestation. E9.5–10.5 SS−/−embryos exhibited severe growth retardation and defective neural tube closure. The lethal phenotype was not rescued by supplementing the dams either with dietary squalene or cholesterol. We speculate that cholesterol is required for the development, particularly of the nervous system, and that the chorioallantoic circulatory system is not mature enough to supply the rapidly growing embryos with maternal cholesterol at this developmental stage.

Early Embryonic Lethality Caused by Targeted Disruption of the 3-Hydroxy-3-methylglutaryl-CoA Reductase Gene

The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr+/–) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr+/– embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr+/– mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr+/– and Hmgcr+/+ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis.

Bcl-2 protein inhibits oxysterol-induced apoptosis through suppressing CPP32-mediated pathway

Oxysterols are presumed to mediate cytotoxicity of oxidized LDL in atherosclerotic lesions. To elucidate its molecular mechanism, we established murine macrophage‐like P388‐D1 cells which over‐express Bcl‐2 protein by retrovirus‐mediated gene transfer. Oxysterols (7‐ketocholesterol, 25‐hydroxycholesterol) induced nuclear condensation and oligonucleosomal DNA fragmentation, which were partially inhibited by Bcl‐2 over‐expression. Though CPP32 inhibitor suppressed the cell death in control cells, it showed no additive protection in the cells over‐expressing Bcl‐2. These findings indicate that oxysterols induce apoptosis via Bcl‐2‐inhibitable and ‐uninhibitable pathways, and the former depends on CPP32 activation.

Induction of macrophage colony-stimulating factor receptor (c-fms) expression in vascular medial smooth muscle cells treated with heparin binding epidermal growth factor-like growth factor.

Vascular smooth muscle cells migrate, proliferate, and transform to foam cells during the atherosclerotic process. We have reported that smooth muscle cells derived from the intima of atherosclerotic lesions express the proto-oncogene c-fms and a scavenger receptor, which are not normally expressed in normal medial smooth muscle cells. In the present study, we demonstrated that heparin binding epidermal growth factor-like growth factor (HB-EGF) induced the expression of c-fms and the scavenger receptor in normal human medial smooth muscle cells to the level observed in the intima. The expression of c-fms was partially inhibited by a protein kinase C inhibitor, suggesting that HB-EGF induces c-fms via pathways that are both dependent on and independent of protein kinase C. By contrast, most of the scavenger receptor induction by HB-EGF was suppressed by protein kinase C inhibitors. These results indicate that two characteristic genes of monocyte-derived macrophages were induced by HB-EGF via different mechanisms. The alteration of gene expression in response to HB-EGF may play an important role in the phenotypic change of smooth muscle cells to macrophage-like foam cells during the atherosclerotic process.

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 –

BACKGROUNDnThe 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).nnnMETHODSANDRESULTSnThis 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.nnnCONCLUSIONSnIn 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).

Apoptotic cell death in atherosclerotic plaques of hyperlipidemic knockout mice.

In an attempt to understand the roles of apoptosis in the development of atherosclerosis, we classified lesions developed in the aortas of apo E- and LDL receptor-deficient mice, murine models of atherosclerosis, and determined frequency, spatial distribution and cell types of apoptotic cells in each lesion. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and nuclear staining with propidium iodide were used to demonstrate apoptotic cells. Mean frequencies of TUNEL-positive cells were as follows: 0%, in Type I, 0.3% in Type II, 0.05% in Type III, 0.06% in Type IV and 0.06% in Type V lesions. Most of the TUNEL-positive cells were filled with fat and distributed in close proximity to lipid pools. The TUNEL-positive cells in the intimal side of the lipid cores were macrophages, while some of those in the adventitial side were smooth muscle cells. In conclusion, apoptosis is involved in the active turn-over of foam cells of both macrophage- and smooth muscle cell-lineage especially in the early atherosclerotic lesions of the hyperlipidemic mice.

The LDL receptor is the major pathway for β-VLDL uptake by mouse peritoneal macrophages

In order to determine the contribution of the low density lipoprotein receptor (LDL-R) to the removal of apoB-containing native lipoproteins by macrophages, we compared the uptake of beta-VLDL in peritoneal macrophages (MPM) from wild type mice and mice lacking the LDL-R. The d<1.006 g/ml lipoproteins obtained from apoE deficient mice fed a high fat diet were poorly degraded by macrophages and caused only a slight formation of CE in macrophages from both types of mice. On the other hand, d<1.006 g/ml lipoproteins obtained from LDL-R deficient mice fed a high fat diet, beta-VLDL with apoE, were avidly taken up by and markedly stimulated CE formation in wild type macrophages, but not in macrophages lacking the LDL-R. The degradation of 125I-labeled-apoE-containing beta-VLDL by wild type MPM was poorly inhibited by unlabeled human LDL, and beta-VLDL without apoE had no effects. In conclusion, we propose that the in vitro uptake of native apoE-enriched lipoproteins by murine macrophages is primarily mediated by the LDL receptor and not by other apoE-recognizing receptor systems such as: the LDL receptor related protein, the VLDL receptor or the triglyceride-rich lipoprotein receptor.

Cholesterol lowering in low density lipoprotein receptor knockout mice overexpressing apolipoprotein E.

Apo E is a key molecule in the lipoprotein metabolism; thus, genetic manipulation of apo E may prove useful in the treatment of hypercholesterolemia. To test the feasibility of this idea, we have generated low density lipoprotein receptor (LDLR) knockout mice that overexpress the rat apo E transgene (ETg+/+:LDLRKO), and compared their plasma lipoprotein profiles with those of nonexpressing LDLR knockout mice (ETg-/-:LDLRKO). On a normal chow diet, the mean plasma cholesterol level of ETg+/+:LDLRKO mice was significantly lower than that of ETg-/-:LDLRKO mice (189 versus 240 mg/dl, P < 0. 01). The LDL fraction was selectively reduced in the ETg+/+:LDLRKO mice. Despite the challenge with an atherogenic diet, cholesterol lowering was persistently observed and fatty streak lesions in the aortic sinus were significantly suppressed in the mice overexpressing apo E. These results imply that stimulation of hepatic production of apo E may be used as a promising adjunctive therapy for homozygous familial hypercholesterolemia.

Effects of treatment with liraglutide on oxidative stress and cardiac natriuretic peptide levels in patients with type 2 diabetes mellitus

Diabetes mellitus (DM) is a risk factor for cardiovascular disease (CVD) [1]. Both traditional and nontraditional molecules have been explored to explain the development of DM-related CVD. Molecules associated with the circulatory system, such as the natriuretic peptides, which are counterregulatory hormones involved in the regulation of volume homeostasis and cardiovascular remodeling, are an important clue for understanding the underlying mechanisms of DM-related CVD [2]. The plasma brain natriuretic peptide (BNP) level is thus a predictor of CVD events and death [3]. Additionally, the oxidative stress burden may in part explain the link between DM and CVD [4]. Although several markers are applicable, the diacron reactive oxygen metabolites (d-ROMs) test and malondialdehyde (MDA) are indicators of the global oxidative stress burden in a clinical setting [5], especially in patients with type 2 DM (T2DM) [6, 7]. n nLiraglutide, a long-acting glucagon-like receptor peptide (GLP)-1 analogue, is a recent treatment modality that may tolerably improve glycemic control [8–10] and provide beneficial cardiovascular effects via extrapancreatic mechanisms [11, 12]. Clinical studies of the association between liraglutide and cardiovascular risk markers, especially nontraditional markers, have been limited. The aim of the present study was to investigate the effects of 24xa0weeks of treatment with liraglutide on cardiovascular risk markers such as BNP, d-ROMs, and MDA in patients with T2DM.

Identification of human ELOVL5 enhancer regions controlled by SREBP

Fatty acid elongase 5 (ELOVL5) is an enzyme involved in the synthesis of polyunsaturated fatty acids. Sterol Regulatory Element-binding Protein (SREBP)-1 activates ELOVL5 and increases polyunsaturated fatty acid synthesis, which in turn negatively affects SREBP-1 expression. Thus, ELOVL5 has been established as an SREBP-1 target gene and an important component of the negative feedback loop of de novo lipogenesis. However, the human ELOVL5 promoter/enhancer has not been fully analyzed and the location of SREBP biding sites around the ELOVL5 gene has yet to be defined. Here we performed a detailed promoter/enhancer analysis of human ELOVL5 gene, and identified two new SREBP binding sites, one in the 10 kb upstream region and one in the exon 1. These two SRE motifs are conserved among mammals and the mechanism found in the present study by which SREBP activates ELOVL5 is considered to be common in mammals. Through these findings, we clarified the molecular mechanism how SREBP activates ELOVL5, an important regulator of de novo lipogenesis.