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.

Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice.

The distribution of human low density lipoprotein (LDL) receptors was studied by immunofluorescence and immunoelectron microscopy in epithelial cells of transgenic mice that express high levels of receptors under control of the metallothionein-I promoter. In hepatocytes and intestinal epithelial cells, the receptors were confined to the basal and basolateral surfaces, respectively. Very few LDL receptors were present in coated pits or intracellular vesicles. In striking contrast, in the epithelium of the renal tubule the receptors were present on the apical (lumenal) surface where they appeared to be concentrated at the base of microvilli and were abundant in vesicles of the endocytic recycling pathway. Intravenously administered LDL colloidal gold conjugates bound to the receptors on hepatocyte microvilli and were slowly internalized, apparently through slow migration into coated pits. We conclude that (a) sorting of LDL receptors to the surface of different epithelial cells varies with each tissue; and (b) in addition to a signal for clustering in coated pits, the LDL receptor may contain a signal for retention in noncoated membrane that is manifest in hepatocytes and intestinal epithelial cells, but not in renal epithelial cells or cultured human fibroblasts.

Cytoplasmic sequence required for basolateral targeting of LDL receptor in livers of transgenic mice.

When expressed in livers of transgenic mice, the human low density lipoprotein (LDL) receptor is specifically targeted to the basolateral (sinusoidal) surface of hepatocytes as determined by immunofluorescence and immunoelectron microscopy. The COOH-terminal cytoplasmic domain of the receptor (residues 790-839) contains a signal for this targeting. A mutant receptor truncated at residue 812 was localized exclusively to the apical (bile canalicular) surface. A mutant receptor terminating at residue 829 showed the normal basolateral distribution, as did a receptor in which alanine was substituted for serine 833, which was previously shown to be a site for phosphorylation in vitro. These data localize the basolateral targeting signal to the 17-residue segment between residues 812 and 828. A 10-amino acid stretch within this segment shows a 4/10 match with a sequence within a previously identified basolateral sorting motif for the receptor for polymeric IgA/IgM in MDCK cells. The four shared residues are spaced at intervals of three, raising the possibility that they all face the same side of an alpha-helix. We conclude that this 10-amino acid stretch may contain a signal that directs certain proteins, including the LDL receptor and the polymeric IgG/IgM receptor, to the basolateral surface of polarized epithelia.

Diet-induced hypercholesterolemia in mice: prevention by overexpression of LDL receptors.

The current studies were designed to determine whether chronic overexpression of low density lipoprotein (LDL) receptors in the liver would protect mice from the increase in plasma LDL-cholesterol that is induced by high-fat diets. A line of transgenic mice was studied that express the human LDL receptor gene in the liver under control of the transferrin promoter. When fed a diet containing cholesterol, saturated fat, and bile acids for 3 weeks, the transgenic mice, in contrast to normal mice, did not develop a detectable increase in plasma LDL. The current data indicate that unregulated overexpression of LDL receptors can protect against diet-induced hypercholesterolemia in mice.

Stimulation of cholesteryl ester synthesis in mouse peritoneal macrophages by cholesterol-rich very low density lipoproteins from the Watanabe heritable hyperlipidemic rabbit, an animal model of familial hypercholesterolemia.

Cholesterol-rich very low density lipoproteins (VLDL) from the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbit induced marked cholesteryl ester accumulation in mouse peritoneal macrophages. This WHHL rabbit, an animal model of human familial hypercholesterolemia, has severe hypercholesterolemia, cutaneous xanthomas, and fulminant atherosclerosis due to the deficiency of the low density lipoprotein (LDL) receptor. When incubated with mouse peritoneal macrophages, the VLDL from WHHL rabbit (WHHL-VLDL) stimulated cholesteryl [14C]oleate synthesis 124-fold more than did VLDL from the normal Japanese White rabbit (control-VLDL). The enhancement in cholesteryl ester synthesis and accumulation of WHHL-VLDL was due to the presence of a high affinity binding receptor site on the macrophage cell surface that mediated the uptake and lysosomal degradation of WHHL-VLDL. Competition studies showed that the uptake and degradation of 125I-WHHL-VLDL was inhibited by unlabeled excess WHHL-VLDL and beta-migrating VLDL (beta-VLDL), but not LDL. Furthermore, the degradation of WHHL-VLDL was not blocked by either fucoidin, polyinosinic acid, or polyguanylic acid, potent inhibitors of the acetylated (acetyl)-LDL binding site, or by acetyl-LDL. These results suggest that macrophages possess a high affinity receptor that recognizes the cholesterol-rich VLDL present in the plasma of the WHHL rabbit and that the receptor which mediates ingestion of WHHL-VLDL seems to be the same as that for beta-VLDL and leads to cholesteryl ester deposition within macrophages. Thus the uptake of the cholesterol-rich VLDL from the WHHL rabbit by macrophages in vivo may play a significant role in the pathogenesis of atherosclerosis in the WHHL rabbit.

Stimulated arachidonate metabolism during foam cell transformation of mouse peritoneal macrophages with oxidized low density lipoprotein.

Changes in arachidonate metabolism were examined in mouse peritoneal macrophages incubated with various types of lipoproteins. Oxidized low density lipoprotein (LDL) was incorporated by macrophages and stimulated macrophage prostaglandin E2 (PGE2) and leukotriene C4 syntheses, respectively, 10.8- and 10.7-fold higher than by the control. Production of 6-keto-PGF1 alpha, a stable metabolite of prostacyclin, was also stimulated. No stimulation was found with native LDL, which was minimally incorporated by the cells. Acetylated LDL and beta-migrating very low density lipoprotein (beta-VLDL), though incorporated more efficiently than oxidized LDL, also had no stimulatory effect. When oxidized LDL was separated into the lipoprotein-lipid peroxide complex and free lipid peroxides, most of the stimulatory activity was found in the former fraction, indicating that stimulation of arachidonate metabolism in the cell is associated with uptake of the lipoprotein-lipid peroxide complex. These results suggest that peroxidative modification of LDL could contribute to the progression of atheroma by stimulating arachidonate metabolism during incorporation into macrophages.

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.

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.

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.

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.

Regulation of apolipoprotein B secretion in hepatocytes from Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia.

Regulation of apolipoprotein B-100 (apo B) secretion in the liver in familial hypercholesterolemia (FH) remains largely unknown. In a previous study, we developed a rabbit hepatocyte culture system and investigated a cellular mechanism which regulates apo B secretion from hepatocytes in response to cellular cholesteryl ester contents [18]. Using this system, in the present study, we investigated regulation of apo B secretion in hepatocytes from the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for FH. Incubation with low density lipoproteins (LDL) at concentrations of 50 or 200 micrograms/ml, which increased both cellular cholesteryl ester and apo B secretion significantly in normal rabbit hepatocytes, did not cause such increases in WHHL rabbit hepatocytes. However, when WHHL rabbit hepatocytes were incubated with LDL at a concentration of 500 micrograms/ml, a significant increase in cellular cholesteryl ester and apo B secretion was observed. The effect of the increase in cellular level of cholesteryl ester upon apo B secretion in WHHL rabbit hepatocytes was compatible with that in normal rabbit hepatocytes. Indeed, when WHHL rabbit hepatocytes were incubated with LDL at 1680 micrograms/ml, a concentration comparable to that of WHHL rabbit plasma, the amount of LDL degradation, cellular cholesteryl ester level, and level of apo B secretion were the same as those in normal rabbit hepatocytes that were incubated with LDL at 160 micrograms/ml, a concentration comparable to that of normal rabbit plasma. In summary, our present study suggests that, at a steady state with such a high plasma concentration of LDL, the hepatic cholesterol content in WHHL rabbits could be set at the same level as in normal rabbits. It was also shown that cellular cholesteryl ester contents had the same regulatory effect on apo B secretion in WHHL rabbit hepatocytes as in normal rabbit hepatocytes. Therefore, we conclude that in the presence of the genetic defect of the LDL receptor, plasma cholesterol in WHHL rabbit is maintained at a concentration such that apo B secretion is similar to that in normal rabbit.

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.

Prevention of atherosclerotic progression in Watanabe rabbits by probucol.

The foam cell has been recognized as a characteristic feature of xanthomas in skin and tendons, and also of atheromas. Many foam cells in these lesions share properties characteristic of the macrophages. Therefore macrophages may be the progenitor of certain foam cells that are involved in atherogenesis. Several investigators demonstrated in vitro that macrophages can ingest large amounts of certain chemically modified lipoproteins, such as acetylated low-density lipoprotein (LDL) and malondialdehyde-treated LDL, through the process of receptor-mediated endocytosis. By this process, macrophages become foam cells. But this process has not been demonstrated in vivo. Recently, oxidized LDL has been suggested to play an important role in atherogenesis by facilitating the accumulation of lipids in macrophages in vitro. Probucol, originally developed as an antioxidant, prevents this oxidative modification of LDL in vitro. Moreover, there are some clinical reports that probucol induces regression of cutaneous and tendon xanthomas in patients with homozygous familial hypercholesterolemia. A question was posed whether in vivo probucol could prevent the progression of atherosclerosis in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia. At age 2 months, 8 WHHL rabbits were classified into 2 groups: group A rabbits were controls and group B rabbits were treated with 1% probucol. After 6 months of treatment, average plasma concentrations of cholesterol were 704 +/- 121 mg/dl in group A and 584 +/- 61 mg/dl in group B. The percentage of surface area of total thoracic aorta with visible plaques in group A vs group B was 54.2 +/- 18.8% vs 7.0 +/- 6.3%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of endothelial platelet-derived growth factor-B-chain and intercellular adhesion molecule-1 by lysophosphatidylcholine.

Lysophosphatidylcholine (lyso-PC) is a major phospholipid component of atherogenic lipoproteins. Lyso-PC has been shown to differentially upregulate the adhesion molecules, such as VCAM-1 and ICAM-1, as well as smooth muscle growth factors, such as PDGF-A, B chains and HB-EGF gene expression in various cultured endothelial cells. In this paper, we demonstrate increased expression of cell- and matrix-associated forms of PDGF-B protein elicited by lyso-PC and further characterized potential signal transduction mechanisms responsible for lyso-PC-induced human umbilical vein endothelial cell. Cycloheximide inhibited PDGF-B but not ICAM-1 mRNA induction by lyso-PC, suggesting the dependence on de novo protein synthesis for PDGF-B, but not ICAM-1. A protein kinase C (PKC) inhibitor did not block lyso-PC-induced increases in PDGF-B or ICAM-1 mRNA. The elevated level of cAMP blocked both PDGF-B and ICAM-1 upregulation by lyso-PC. However cAMP-elevating agents did not suppress ICAM-1 upregulation by PMA. Taken together, PDGF-B and ICAM-1 gene induction by lyso-PC may involve different signaling mechanisms; however, both appear to be independent of PMA-regulatable PKC activation but are suppressed by increased levels of intracellular cAMP.

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.

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.

Coexistent myelodysplastic syndrome and non-Hodgkin lymphoma. Report of a case and review of the literature.

The patients with a hematopoietic neoplasm can develop myelodysplastic syndrome following chemotherapy. We report herein a case of non-Hodgkin B-cell lymphoma and refractory anemia with ringed sideroblasts. Absence of the preceding chemotherapy establishes the true coincidence of the two disorders. We review 25 similar cases reported in the last 11 years and discuss the possible etiology of coexistence.

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.

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.