Olmesartan, a novel angiotensin II type 1 receptor antagonist, reduces severity of atherosclerosis in apolipoprotein E deficient mice associated with reducing superoxide production.

BACKGROUND AND AIM: Oxidative stress may play an important role in the development of atherosclerosis. Some angiotensin II type 1 (AT(1)) receptor antagonists have the capacity of reducing oxidative stress in addition to the hemodynamic actions. Accordingly, we assessed the hypothesis that olmesartan, a novel AT(1) receptor antagonist, reduced the severity of atherosclerosis in apolipoprotein (apo) E-deficient mice associated with reducing oxidative stress. METHODS AND RESULTS: Atherosclerosis was induced in apo E-deficient mice fed a high fat diet. Mice were intraperitoneally treated with an injection of olmesartan (1mg/kg/day) daily over 8 weeks, and were compared with the untreated controls. Blood pressure was not changed significantly by the olmesartan treatment. Fatty streak plaque developed in apo E-deficient mice, and was suppressed in mice that received olmesartan. In addition, olmesartan reduced not only superoxide production but the overload of oxidative stress in aortic walls. There were no significant differences in serum lipid levels between olmesartan-treated and -untreated groups. In vitro study showed that both olmesartan and its active metabolite RNH-6270, an enantiomer of olmesartan, suppressed interferon-γ, macrophage inflammatory protein-2, and thioredoxin (a marker of oxidative stress) concentrations in cultured cells. CONCLUSION: Olmesartan may suppress atherosclerosis via reducing not only superoxide production but also the overload of oxidative stress in this animal model.

Plasma ghrelin levels in healthy elderly volunteers: the levels of acylated ghrelin in elderly females correlate positively with serum IGF-I levels and bowel movement frequency and negatively with systolic blood pressure.

Aging is associated with a decrease in growth hormone (GH) secretion, appetite and energy intake. As ghrelin stimulates both GH secretion and appetite, reductions in ghrelin levels may be involved in the reductions in GH secretion and appetite observed in the elderly. However, only preliminary studies have been performed on the role of ghrelin in elderly subjects. In this study, we sought to clarify the physiologic implications of the age-related alterations in ghrelin secretion by determining plasma ghrelin levels and other clinical parameters in healthy elderly subjects. Subjects were > or = 65 years old, corresponding to the SENIEUR protocol, had not had a resection of the upper gastrointestinal tract and had not been treated with hormones. One hundred and five volunteers (49 men and 56 women) were admitted to this study (73.4 +/- 6.3 years old). Plasma levels of acylated ghrelin in elderly female subjects positively correlated with serum IGF-I levels and bowel movement frequency and negatively with systolic blood pressure. In elderly men, desacyl ghrelin levels correlated only weakly with bowel movement frequency. These findings suggest that the plasma levels of the acylated form of ghrelin may influence the age-related alterations in GH/IGF-I regulation, blood pressure and bowel motility. These observational associations warrant further experimental studies to clarify the physiologic significance of these effects.

Functional blockade of platelet-derived growth factor receptor-beta but not of receptor-alpha prevents vascular smooth muscle cell accumulation in fibrous cap lesions in apolipoprotein E-deficient mice.

BACKGROUND: The vascular smooth muscle cell (VSMC) is the central cell component involved in the fibroproliferative response in atherogenesis. As the lesion advances, VSMCs migrate from the media into the subendothelial space, thereby forming fibrous plaque lesions. Platelet-derived growth factor (PDGF) has been known to be a potent chemoattractant and mitogen for SMCs, but the pathophysiological role of the 2 PDGF receptors, receptor-alpha (PDGFR-alpha) and receptor-beta (PDGFR-beta) in atherogenesis is poorly understood. To clarify this problem, we prepared antagonistic rat monoclonal antibodies, APA5 and APB5, against murine PDGFR-alpha and PDGFR-beta, respectively. METHODS AND RESULTS: Apolipoprotein E-deficient mice were fed a high-fat diet containing 0.3% cholesterol from 6 weeks of age and subjected to injection with 1 mg/d IP of either antibody from 12 to 18 weeks every other day. In the mice injected with APB5, the aortic atherosclerotic lesion size and the number of intimal VSMCs were reduced by 67% and 80%, respectively, compared with the control mice injected with irrelevant rat IgG. In contrast, the mice that received APA5 showed only minimal reduction of lesion size, and a large number of VSMCs were observed in the intima. In the intima of advanced lesions, APB5 immunolabeled VSMCs, whereas APA5 could detect VSMCs mainly in the media. CONCLUSIONS: These results indicate that PDGFR-beta plays a significant role in formation of fibrous atherosclerotic lesions and that regulation of the signal transduction through PDGFR-beta could affect atherogenesis in mice.

Expression of basic helix-loop-helix proteins in the glomeruli.

BACKGROUND: Basic helix loop helix (bHLH) proteins play a critical role in the differentiation of not only striated muscle cells but also adipocytes, neuron cells and smooth muscle cells. Previous studies have established in vitro mouse mesangial cells (MCs) to maintain the differentiated smooth muscle phenotype. MATERIALS AND METHODS: The purpose of the present study was to clone bHLH proteins from these MCs using the primers designed from a homologous sequence specific to bHLH, and to analyze the presence of bHLH proteins in normal kidney in vivo. From the cloning of MCs in vitro, we identified myf5 and herculin mRNA but not myoD. The expression of bHLH proteins in vivo was examined by immunohistochemistry with each specific antibody. RESULTS: The MCs in newborn mice possessed Id but did not express either protein herculin or myoD. On the other hand, mature MCs expressed both myf5 and herculin. The Id protein disappeared in mature glomeruli. CONCLUSION: These results suggest that bHLH proteins are an important factor for mature MCs in vivo.

Role of oxidized LDL in atherosclerosis.

A critical event in the early stages of atherosclerosis is the focal accumulation of lipid-laden foam cells derived from macrophages. In various cholesterol-fed animal models of atherosclerosis, localized attachment of circulating monocytes to arterial endothelial cells appeared to precede the formation of foam cells. It is suggested that monocyte recruitment into early lesions depends on the endothelial adhesiveness for monocytes and lymphocytes. In vivo and in vitro experiments have identified molecules, such as ICAM-1, VCAM-1, and P-selectin, that can support the adhesion of monocytes and lymphocytes. Moreover, oxidized LDL, lysophosphatidyl-choline, and oxidized fatty acids induce the expression not only of these adhesion molecules but also of scavenger receptors, such as CD-36, SR-A, and LOX-1. Recently, we isolated and characterized the novel receptors for oxidized LDL, namely, LOX-1 and SR-PSOX. Expression of LOX-1 is found on endothelial cells, smooth muscle cells, and macrophages, whereas SR-PSOX is expressed on macrophages. In this paper the significance of oxidized LDL and its receptors, LOX-1 and SR-PSOX, in terms of atherogenesis is discussed.

Study on PDGF receptor beta pathway in glomerular formation in neonate mice.

The assembly of vascular endothelial cells (ECs) and smooth muscle cells is a critical event in the development of the cardiovascular system. Although the role of ECs in this event has been studied intensively, the cross-talk between the two cell components remains poorly understood. In this study, we blocked platelet-derived growth factor receptor (PDGFR) pathways in mice by antagonistic rat monoclonal antibody APB5 against murine PDGFR-beta and examined glomerular capillary formation.

[Advance in clinical studies on atherosclerosis].

Atherosclerosis leading to coronary heart disease and cerebrovascular disorders is the most serious cause of death in the industrialized societies. For the last two decades, great advances have been made in understanding the pathogenesis of those disorders by studies based on molecular and cell biology. These findings have been confirmed by several randomized clinical trials. In this article, the current status of treatment and prevention of atherosclerosis will be reviewed and discussed.

Oxidized-LDL and atherosclerosis. Role of LOX-1.

The accumulation of substantial numbers of monocyte/macrophages and activated T lymphocytes in focal areas of the arterial intima appears to be a hallmark of atherosclerosis. Our report demonstrated that lysophosphatidylcholine (lyso-PC), a polar phospholipid component that is increased in atherosclerotic lipoproteins, such as oxidized LDL and remnant lipoproteins in diabetic and Type 3 hyperlipidemia, can upregulate adhesion molecules for monocytes and T lymphocytes, and growth factors, such as heparin-binding epidermal growth factor-like growth factor and PDGF A and B chains. Recently, we identified the novel receptor for oxidized LDL, named LOX-1. We summarize the importance of the interaction between oxidized LDL and its receptor, LOX-1, in terms of early stage atherogenesis.

Overexpression of low density lipoprotein receptor eliminates apolipoprotein B100-containing lipoproteins from circulation and markedly prevents early atherogenesis in apolipoprotein E-deficient mice.

Apolipoprotein E (ApoE) plays a pivotal role in the metabolism of apolipoprotein B (apoB)-containing lipoproteins. The defective apoE gene in humans can cause elevated plasma levels of apoB-containing lipoproteins such as chylomicron remnant and intermediate density lipoprotein (IDL). In this study, we examined whether liver-selective high-level expression of low-density lipoprotein receptor (LDLR) could affect the lipoprotein profile and atherogenesis in apoE-deficient (apoE-/-) mice. ApoE knockout mice expressing LDLR transgene in liver [apoE-/-;Tg(LDLR+/-)] were prepared after mating apoE-/- mice with the human LDLR transgenic mice. The apoE-/-;Tg(LDLR+/-) and littermate apoE-/- mice were fed a normal diet and sacrificed at 18 weeks of age. (1) The plasma levels of cholesterol and triglyceride in apoE-/-;Tg(LDLR+/-) mice were 51 and 33% lower than those of apoE-/- mice, respectively. (2) In the plasma of apoE-/-;Tg(LDLR+/-) mice, the levels of apoB-containing lipoprotein were reduced and apoB100-containg particles were totally eliminated. (3) By histochemical analysis, apoE-/-;Tg(LDLR+/-) mice showed drastic suppression of early atherogenesis; the lesion area of these mice was 1/70 of that in the littermate apoE-/- mice. These results indicate that, even in apoE-defective subjects, induction of hepatic LDLR expression could protect from early atherogenesis.

A novel snail-related transcription factor Smuc regulates basic helix-loop-helix transcription factor activities via specific E-box motifs.

Snail family proteins are zinc finger transcriptional regulators first identified in Drosophila which play critical roles in cell fate determination. We identified a novel Snail -related gene from murine skeletalmusclecells designated Smuc. Northern blot analysis showed that Smuc was highly expressed in skeletal muscle and thymus. Smuc contains five putative DNA-binding zinc finger domains in its C-terminal half. In electrophoretic mobility shift assays, recombinant zinc finger domains of Smuc specifically bound to CAGGTG and CACCTG E-box motifs (CANNTG). Because basic helix-loop-helix transcription factors (bHLH) bind to the same E-box sequences, we examined whether Smuc competes with the myogenic bHLH factor MyoD for DNA binding. Smuc inhibited the binding of a MyoD-E12 complex to the CACCTG E-box sequence in a dose-dependent manner and suppressed the transcriptional activity of MyoD-E12. When heterologously targeted to the thymidine kinase promoter as fusion proteins with the GAL4 DNA-binding domain, the non-zinc finger domain of Smuc acted as a transcriptional repressor. Furthermore, overexpression of Smuc in myoblasts repressed transactivation of muscle differentiation marker Troponin T. Thus, Smuc might regulate bHLH transcription factors by zinc finger domains competing for E-box binding, and non-zinc finger repressor domains might also confer transcriptional repression to control differentiation processes.

Oxidized LDL and expression of monocyte adhesion molecules.

Accumulation of substantial numbers of monocyte/macrophages, as well as activated T lymphocytes, in focal areas of arterial intima appears to be a hallmark of atherogenesis. Our report demonstrated that lysophosphatidylcholine (lyso-PC), a polar phospholipid component that is increased in atherogenic lipoproteins, such as oxidized LDL and remnants lipoproteins in diabetic and type III hyperlipidemic patients, can upregulate adhesion molecules for monocytes and T lymphocytes, and growth factors, such as heparin-binding epidermal growth factor-like growth factor and PDGF-A and B chains. Recently we identified the novel receptor for oxidized LDL, named Lox-1. Therefore in this paper we summarize the importance of the interaction between oxidized LDL and its receptor, LOX-1 in terms of early stage of atherogenesis.

[Gene polymorphisms in atherosclerotic diseases and lipoprotein metabolism disorders].

Atherosclerosis results from complex inflammatory-fibroproliferative responses. Among the factors that could affect atherogenesis, the genes involved in regulation of the plasma lipoprotein levels are believed to play central roles. This paper focuses on the genes regulating the metabolism of the three major atherogenic lipoproteins, i.e. LDL, remnants, and Lp (a), and discusses the clinical significance of studying their polymorphism.

[Aspergillosis following influenza A infection].

An 83-year-old man had an influenza-like upper respiratory infection that progressed to pneumonia and respiratory insufficiency during a period two weeks. After admission, anti-influenza A antibody increased 32-fold and antibiotic treatment had little effect on the pneumonia. Aspergillus antigen was detected from his serum and pleural effusion, however, culture of sputum was negative for aspergillus. Administration of amphotericin B reduced the serum level of aspergillus antigen, however he died due to the progression of respiratory insufficiency and bloody sputum. Aspergillus infection is generally thought to occur in immunocompromised hosts, but this patient had no apparent immunosuppressive conditions except for his age before the influenza A infection. His WBC and lymphocyte count temporally decreased to 2,000 x 10(6)/L (lymphocytes 160 x 10(6)/L) followed by aspergillus infection. This temporally reduction of lymphocytes is thought to have been responsible for the aspergillus infection. Complications of influenza infection are sometimes fatal and vaccination against influenza seems necessary in high risk individuals such as elderly people.

Compensated endocytosis of LDL by hamster cells co-expressing the two distinct mutant LDL receptors defective in endocytosis and ligand binding.

The low density lipoprotein receptor (LDLR) regulates the plasma cholesterol level by mediating endocytosis of LDL. We established stable hamster cell lines expressing two LDLRs with distinct functional defects, i.e., endocytosis and ligand binding. In the cell line expressing only I189D h/r (human-rat chimeric) LDLR, defective in LDL binding, very little amount of LDL was internalized, although the receptor was endocytosed efficiently. In the cell line expressing Y807C LDLR solely, very few receptors were located in coated pits or endocytosed, while LDL binding to the receptor was not disrupted. In striking contrast, in the cells co-expressing both receptors, a much larger number of Y807C LDLR were internalized and co-located with I189D h/r LDLR in the perinuclear region. In these cells, LDL was bound exclusively to Y807C LDLR and its uptake was enhanced by 80% as compared to the cell expressing Y807C LDLR solely, whereas LDL binding affinity was not changed. These results suggest that a defect of the essential motif for endocytosis, cysteine 807, could be compensated by co-expression of I189D h/r LDLR, but the LDL binding was not affected.

Intraperitoneal administration of anti-c-fms monoclonal antibody prevents initial events of atherogenesis but does not reduce the size of advanced lesions in apolipoprotein E-deficient mice.

BACKGROUND: Atherosclerosis results from complex inflammatory-fibroproliferative responses. To elucidate the central role of macrophage and macrophage-colony stimulating factor (M-CSF) during atherogenesis, we used a new strategy to administer to adult apolipoprotein E (apoE)-deficient mice a monoclonal antibody (AFS98) raised against c-fms, the receptor of M-CSF. METHODS AND RESULTS: When 6-week-old apoE-deficient mice were fed a high-fat diet and injected with 2 mg of AFS98 intraperitoneally on alternate days for 6 weeks, accumulation of macrophage-derived foam cells in the aortic root was suppressed by 70% compared with that in controls. This preventive effect was associated with neither remarkable decrease of the number of circulating monocytes nor systemic growth retardation. In contrast, when apoE-deficient mice that had been fed a high-fat diet from 6 weeks of age were given AFS98 from 12 to 18 weeks of age, a minimal protective effect on lesion size was observed. CONCLUSIONS: These results suggest that (1) macrophage and M-CSF/c-fms play an essential role in the arterial wall during development of the fatty streak lesion and (2) blockade of the M-CSF/c-fms pathway could act as protection from at least early atherogenesis but could have a less preventive effect on maintenance of the advanced lesions.

[Endothelial injury by oxidized LDL].

Recent progress in the research of oxidized LDL has revealed that this lipoprotein causes not only foam cell transformation of macrophages but also several endothelial dysfunction, and the effects on endothelial cells are also involved with the process of atherogenesis. Receptors for oxidized LDL on endothelial cells, such as LOX-1 and SREC, have been cloned and their characteristics are now under investigation. In addition to lowering plasma cholesterol level, it is expected that new strategies to prevent atherosclerosis is established by focusing on the endothelial injury caused by oxidized LDL.

Cholesterol efflux effect of high density lipoprotein is impaired by whole cigarette smoke extracts through lipid peroxidation.

It has been reported that high density lipoprotein (HDL) plays an anti-atherogenic role by stimulating cholesterol efflux from the foam cells in the atheromatous lesion. In this study, we prepared a novel modified form of HDL (CS-HDL) by incubating HDL with whole cigarette smoke (CS) extracts containing both particulate matter and gas-phase smoke, and examined its effect on cholesterol efflux. CS-HDL showed a marked increase of conjugated dienes and denaturation of apoA-I, a major protein component of HDL. The cholesterol efflux effect of CS-HDL was remarkably reduced to the same level as that of oxidatively modified HDL induced by copper ion (Ox-HDL). Addition of 20 microg/ml superoxide dismutase (SOD) during the CS-modification of HDL caused retrieval of cholesterol efflux activity by 53% and a remarkable decrease in the conjugated dienes level. SOD, however, had no ameliorative effect on apoA-I denaturation. When HDL was incubated only with gas-phase smoke (gasCS-HDL), neither increase of conjugated dienes nor impairment of the cholesterol efflux effect was observed, whereas apoA-I was denaturated to the same extent as seen in CS-HDL. These results indicate that whole CS-extracts, but not gas-phase smoke, reduces cholesterol efflux effect of HDL and that lipid peroxidation associated with superoxide anion is involved in this functional impairment.