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1.
Proc Natl Acad Sci U S A ; 98(15): 8838-43, 2001 Jul 17.
Article in English | MEDLINE | ID: mdl-11447277

ABSTRACT

Human apolipoprotein (apo) E4, a major risk factor for Alzheimer's disease (AD), occurs in amyloid plaques and neurofibrillary tangles (NFTs) in AD brains; however, its role in the pathogenesis of these lesions is unclear. Here we demonstrate that carboxyl-terminal-truncated forms of apoE, which occur in AD brains and cultured neurons, induce intracellular NFT-like inclusions in neurons. These cytosolic inclusions were composed of phosphorylated tau, phosphorylated neurofilaments of high molecular weight, and truncated apoE. Truncated apoE4, especially apoE4(Delta 272--299), induced inclusions in up to 75% of transfected neuronal cells, but not in transfected nonneuronal cells. ApoE4 was more susceptible to truncation than apoE3 and resulted in much greater intracellular inclusion formation. These results suggest that apoE4 preferentially undergoes intracellular processing, creating a bioactive fragment that interacts with cytoskeletal components and induces NFT-like inclusions containing phosphorylated tau and phosphorylated neurofilaments of high molecular weight in neurons.


Subject(s)
Alzheimer Disease/metabolism , Apolipoproteins E/metabolism , Brain/metabolism , Neurofibrillary Tangles/metabolism , Peptide Fragments/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/pathology , Apolipoprotein E4 , Apolipoproteins E/physiology , Brain/pathology , Cells, Cultured , Cytosol/metabolism , Humans , Inclusion Bodies/metabolism , Inclusion Bodies/pathology , Intracellular Fluid/metabolism , Mutagenesis , Neurofibrillary Tangles/pathology , Neurofilament Proteins/metabolism , Neurons/metabolism , Peptide Fragments/physiology , tau Proteins/metabolism
2.
J Biol Chem ; 275(41): 31770-7, 2000 Oct 13.
Article in English | MEDLINE | ID: mdl-10918055

ABSTRACT

Amyloid Abeta deposition is a neuropathologic hallmark of Alzheimer's disease. Activated microglia are intimately associated with plaques and appear to facilitate Abeta deposition, an event believed to contribute to pathogenesis. It is unclear if microglia can modulate pathogenesis of Alzheimer's disease by secreting lipoprotein particles. Here we show that cultured BV2 murine microglial cells, like astrocytes, secrete apolipoprotein E (apoE) and apolipoprotein J (apoJ) in a time-dependent manner. To isolate and identify BV2 microglial particles, gel filtration chromatography was employed to fractionate BV2-conditioned medium. Analyses by Western blot, lipid determination, electron microscopy, and native gel electrophoresis demonstrate that BV2 microglial cells release spherical low density lipoprotein (LDL)-like lipid-containing particles rich in apoJ but poor in apoE. These microglial particles are dissimilar in size, shape, and lipoprotein composition to astrocyte-derived particles. The microglial-derived particles were tested for functional activity. Under conditions of suppressed de novo cholesterol synthesis, the LDL-like particles effectively rescued primary rat cortical neurons from mevastatin-induced neurotoxicity. The particles were also shown to bind Abeta. We speculate that the LDL-like apoJ-rich apoE-poor microglial lipoproteins preferentially bind the lipoprotein receptor, recognizing apoJ, which is abundant in the choroid plexus, facilitating Abeta clearance from the brain. BV2 cells also secrete an apoE-rich lipid-poor species that binds Abeta. Consistent with the role of apoE in Abeta fibril formation and deposition, this microglial species may promote plaque formation.


Subject(s)
Apolipoproteins E/isolation & purification , Glycoproteins/isolation & purification , Lipoproteins, LDL/chemistry , Liposomes/chemistry , Microglia/chemistry , Molecular Chaperones , Nerve Tissue Proteins/isolation & purification , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Apolipoproteins E/chemistry , Apolipoproteins E/immunology , Apolipoproteins E/ultrastructure , Blotting, Western , Cell Death/drug effects , Cells, Cultured , Chromatography, Gel , Clusterin , Culture Media, Conditioned/chemistry , Electrophoresis, Polyacrylamide Gel , Glycoproteins/chemistry , Glycoproteins/immunology , Glycoproteins/ultrastructure , Kinetics , Lipoproteins, LDL/metabolism , Lipoproteins, LDL/ultrastructure , Liposomes/metabolism , Lovastatin/analogs & derivatives , Lovastatin/pharmacology , Mice , Microglia/cytology , Microglia/metabolism , Microscopy, Electron , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/immunology , Nerve Tissue Proteins/ultrastructure , Neurons/cytology , Neurons/drug effects , Particle Size , Plaque, Amyloid/chemistry , Plaque, Amyloid/metabolism , Protein Binding , Rats
3.
Nat Genet ; 25(1): 87-90, 2000 May.
Article in English | MEDLINE | ID: mdl-10802663

ABSTRACT

Triglycerides (or triacylglycerols) represent the major form of stored energy in eukaryotes. Triglyceride synthesis has been assumed to occur primarily through acyl CoA:diacylglycerol transferase (Dgat), a microsomal enzyme that catalyses the final and only committed step in the glycerol phosphate pathway. Therefore, Dgat has been considered necessary for adipose tissue formation and essential for survival. Here we show that Dgat-deficient (Dgat-/-) mice are viable and can still synthesize triglycerides. Moreover, these mice are lean and resistant to diet-induced obesity. The obesity resistance involves increased energy expenditure and increased activity. Dgat deficiency also alters triglyceride metabolism in other tissues, including the mammary gland, where lactation is defective in Dgat-/- females. Our findings indicate that multiple mechanisms exist for triglyceride synthesis and suggest that the selective inhibition of Dgat-mediated triglyceride synthesis may be useful for treating obesity.


Subject(s)
Acyltransferases/deficiency , Acyltransferases/genetics , Obesity/metabolism , Triglycerides/biosynthesis , Absorption , Animals , Body Temperature Regulation/genetics , Calorimetry , Diacylglycerol O-Acyltransferase , Dietary Fats/administration & dosage , Energy Metabolism/genetics , Female , Insulin Resistance/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/enzymology , Obesity/genetics , Triglycerides/genetics
4.
J Clin Invest ; 105(6): 711-9, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10727439

ABSTRACT

Inhibitors of acyl CoA:cholesterol acyltransferase (ACAT) have attracted considerable interest as a potential treatment for atherosclerosis. Currently available inhibitors probably act nonselectively against the two known ACATs. One of these enzymes, ACAT1, is highly expressed in macrophages in atherosclerotic lesions, where it contributes to foam-cell formation. In this study, we examined the effects of selective ACAT1 deficiency in two mouse models of atherosclerosis. In the setting of severe hypercholesterolemia caused by deficiency in apoE or the LDL receptor (LDLR), total ACAT1 deficiency led to marked alterations in cholesterol homeostasis and extensive deposition of unesterified cholesterol in the skin and brain. Bone marrow transplantation experiments demonstrated that ACAT1 deficiency in macrophages was sufficient to cause dermal xanthomas in hyperlipidemic LDLR-deficient mice. ACAT1 deficiency did not prevent the development of atherosclerotic lesions in either apoE-deficient or LDLR-deficient mice, despite causing relatively lower serum cholesterol levels. However, the lesions in ACAT1-deficient mice were atypical in composition, with reduced amounts of neutral lipids and a paucity of macrophages in advanced lesions. Although the latter findings may be associated with increased lesion stability, the marked alterations in cholesterol homeostasis indicate that selectively inhibiting ACAT1 in the setting of severe hyperlipidemia may have detrimental consequences.


Subject(s)
Arteriosclerosis/etiology , Cholesterol/metabolism , Foam Cells/pathology , Hypercholesterolemia/genetics , Isoenzymes/physiology , Macrophages/enzymology , Sterol O-Acyltransferase/physiology , Xanthomatosis/etiology , Animals , Aorta/pathology , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Apolipoproteins E/physiology , Arteriosclerosis/enzymology , Arteriosclerosis/genetics , Arteriosclerosis/pathology , Bone Marrow Transplantation , Crosses, Genetic , Diet, Atherogenic , Foam Cells/enzymology , Hypercholesterolemia/complications , Hypercholesterolemia/enzymology , Hypercholesterolemia/pathology , Isoenzymes/deficiency , Isoenzymes/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, LDL/deficiency , Receptors, LDL/genetics , Receptors, LDL/physiology , Skin/enzymology , Skin/pathology , Sterol O-Acyltransferase/deficiency , Sterol O-Acyltransferase/genetics , Xanthomatosis/enzymology , Xanthomatosis/genetics , Xanthomatosis/pathology
5.
Am J Pathol ; 156(1): 139-50, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10623661

ABSTRACT

Cerebrovascular amyloid deposition and microvascular degeneration are frequently associated with Alzheimer's disease (AD), but the etiology and pathogenetic role of these abnormalities are unknown. Recently, transforming growth factor-beta1 (TGF-beta1) was implicated in cerebrovascular amyloid formation in transgenic mice with astroglial overproduction of TGF-beta1 and in AD. We tested whether TGF-beta1 overproduction induces AD-like cerebrovascular degeneration and analyzed how cerebrovascular abnormalities develop over time in TGF-beta1-transgenic mice. In cerebral microvessels from 3- to 4-month-old TGF-beta1-transgenic mice, which display a prominent perivascular astrocytosis, levels of the basement membrane proteins perlecan and fibronectin were severalfold higher than in vessels from nontransgenic mice. Consistent with this increase, cortical capillary basement membranes of TGF-beta1 mice were significantly thickened. These changes preceded amyloid deposition, which began at around 6 months of age. In 9- and 18-month-old TGF-beta1 mice, various degenerative changes in microvascular cells of the brain were observed. Endothelial cells were thinner and displayed abnormal, microvilli-like protrusions as well as occasional condensation of chromatin, and pericytes occupied smaller areas in capillary profiles than in nontransgenic controls. Similar cerebrovascular abnormalities have been reported in AD. We conclude that chronic overproduction of TGF-beta1 triggers an accumulation of basement membrane proteins and results in AD-like cerebrovascular amyloidosis and microvascular degeneration. Closely related processes may induce cerebrovascular pathology in AD.


Subject(s)
Alzheimer Disease/pathology , Astrocytes/metabolism , Blood Vessels/pathology , Transforming Growth Factor beta/metabolism , Aging/metabolism , Alzheimer Disease/metabolism , Animals , Basement Membrane/drug effects , Basement Membrane/metabolism , Blood Vessels/drug effects , Blood Vessels/metabolism , Cerebrovascular Circulation , Dose-Response Relationship, Drug , Humans , Mice , Mice, Inbred BALB C , Mice, Transgenic/genetics , Microcirculation/drug effects , Microscopy, Electron , Time Factors , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/pharmacology
6.
Proc Natl Acad Sci U S A ; 95(8): 4544-9, 1998 Apr 14.
Article in English | MEDLINE | ID: mdl-9539774

ABSTRACT

We have generated mice with markedly elevated plasma levels of human low density lipoprotein (LDL) and reduced plasma levels of high density lipoprotein. These mice have no functional LDL receptors [LDLR-/-] and express a human apolipoprotein B-100 (apoB) transgene [Tg(apoB+/+)] with or without an apo(a) transgene [Tg(apoa+/-)]. Twenty animals (10 males and 10 females) of each of the following four genotypes were maintained on a chow diet: (i) LDLR-/-, (ii) LDLR-/-;Tg(apoa+/-), (iii) LDLR-/-;Tg(apoB+/+), and (iv)LDLR-/-;Tg(apoB+/+);Tg(apo+/-). The mice were killed at 6 mo, and the percent area of the aortic intimal surface that stained positive for neutral lipid was quantified. Mean percent areas of lipid staining were not significantly different between the LDLR-/- and LDLR-/-;Tg(apoa+/-) mice (1.0 +/- 0.2% vs. 1.4 +/- 0.3%). However, the LDLR-/-;Tg(apoB+/+) mice had approximately 15-fold greater mean lesion area than the LDLR-/- mice. No significant difference was found in percent lesion area in the LDLR-/-;Tg(apoB+/+) mice whether or not they expressed apo(a) [18.5 +/- 2.5%, without lipoprotein(a), Lp(a), vs. 16.0 +/- 1.7%, with Lp(a)]. Histochemical analyses of the sections from the proximal aorta of LDLR-/-;Tg(apoB+/+) mice revealed large, complex, lipid-laden atherosclerotic lesions that stained intensely with human apoB-100 antibodies. In mice expressing Lp(a), large amounts of apo(a) protein colocalized with apoB-100 in the lesions. We conclude that LDLR-/-; Tg(apoB+/+) mice exhibit accelerated atherosclerosis on a chow diet and thus provide an excellent animal model in which to study atherosclerosis. We found no evidence that apo(a) increased atherosclerosis in this animal model.


Subject(s)
Apolipoproteins B/biosynthesis , Apolipoproteins/biosynthesis , Arteriosclerosis/genetics , Receptors, LDL/deficiency , Animal Feed , Animals , Aorta, Thoracic/pathology , Apolipoprotein B-100 , Apolipoproteins/genetics , Apolipoproteins B/blood , Apolipoproteins B/genetics , Apoprotein(a) , Arteriosclerosis/blood , Arteriosclerosis/pathology , Cholesterol/blood , Crosses, Genetic , Female , Humans , Lipoprotein(a)/blood , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Mice, Transgenic , Receptors, LDL/genetics , Sex Characteristics
7.
J Biol Chem ; 273(5): 3013-20, 1998 Jan 30.
Article in English | MEDLINE | ID: mdl-9446615

ABSTRACT

The goal of this study was to determine whether actin-binding protein (ABP) regulates membrane composition. ABP-deficient and ABP-containing cells were transfected with the cDNAs coding for glycoprotein (GP) Ib-IX, a platelet receptor that interacts with ABP. Most of the GP Ib-IX remained inside the ABP-deficient cells. When ABP was present, functional GP Ib-IX was inserted into the membrane. GP Ib-IX lacking the domain that interacts with ABP also showed increased membrane insertion in ABP-expressing cells. Furthermore, a fragment of ABP that lacks the dimerization and GP Ib-IX-binding sites restored the spreading of the cells and increased the amount of GP Ib-IX in the membrane. Finally, expression of ABP also increased endogenous beta1 integrin in the membrane. These results indicate that 1) ABP maintains the properties of the cell such that adhesion receptors can be efficiently expressed in the membrane; 2) increased receptor expression is accompanied by increased ability of the cell to spread; and 3) ABP exerts its effect by a mechanism that does not appear to involve direct cross-linking of actin filaments or direct interaction with receptors.


Subject(s)
Cell Membrane/physiology , Glycosylphosphatidylinositols/physiology , Microfilament Proteins/physiology , Flow Cytometry , Gene Expression Regulation , Humans , Integrin beta1/metabolism , Microscopy, Phase-Contrast , Transfection , Tumor Cells, Cultured
8.
J Biol Chem ; 273(4): 1896-903, 1998 Jan 23.
Article in English | MEDLINE | ID: mdl-9442022

ABSTRACT

To determine the mechanisms by which human hepatic lipase (HL) contributes to the metabolism of apolipoprotein (apo) B-containing lipoproteins and high density lipoproteins (HDL) in vivo, we developed and characterized HL transgenic mice. HL was localized by immunohistochemistry to the liver and to the adrenal cortex. In hemizygous (hHLTg+/0) and homozygous (hHLTg+/+) mice, postheparin plasma HL activity increased by 25- and 50-fold and plasma cholesterol levels decreased by 80% and 85%, respectively. In mice fed a high fat, high cholesterol diet to increase endogenous apoB-containing lipoproteins, plasma cholesterol decreased 33% (hHLTg+/0) and 75% (hHLTg+/+). Both apoB-containing remnant lipoproteins and HDL were reduced. To extend this observation, the HL transgene was expressed in human apoB transgenic (huBTg) and apoE-deficient (apoE-/-) mice, both of which have high plasma levels of apoB-containing lipoproteins. (Note that the huBTg mice that were used in these studies were all hemizygous for the human apoB gene.) In both the huBTg,hHLTg+/0 mice and the apoE-/-,hHLTg+/0 mice, plasma cholesterol decreased by 50%. This decrease was reflected in both the apoB-containing and the HDL fractions. To determine if HL catalytic activity is required for these decreases, we expressed catalytically inactive HL (HL-CAT) in apoE-/- mice. The postheparin plasma HL activities were similar in the apoE-/- and the apoE-/-,HL-CAT+/0 mice, reflecting the activity of the endogenous mouse HL and confirming that the HL-CAT was catalytically inactive. However, the postheparin plasma HL activity was 20-fold higher in the apoE-/-,hHLTg+/0 mice, indicating expression of the active human HL. Immunoblotting demonstrated high levels of human HL in postheparin plasma of both apoE-/-,hHLTg+/0 and apoE-/-,HL-CAT+/0 mice. Plasma cholesterol and apoB-containing lipoprotein levels were approximately 60% lower in apoE-/-,HL-CAT+/0 mice than in apoE-/- mice. However, the HDL were only minimally reduced. Thus, the catalytic activity of HL is critical for its effects on HDL but not for its effects on apoB-containing lipoproteins. These results provide evidence that HL can act as a ligand to remove apoB-containing lipoproteins from plasma.


Subject(s)
Apolipoproteins B/metabolism , Lipase/metabolism , Lipoproteins, HDL/metabolism , Animals , Apolipoproteins E/metabolism , Blotting, Western , Catalysis , Female , Humans , Lipase/genetics , Lipids/blood , Lipoproteins/blood , Liver/enzymology , Mice , Mice, Transgenic , RNA, Messenger/metabolism , Tissue Distribution
9.
J Biol Chem ; 272(36): 22685-94, 1997 Sep 05.
Article in English | MEDLINE | ID: mdl-9312550

ABSTRACT

Transgenic rabbits were produced that expressed high plasma levels (30-70 mg/dl) of human apolipoprotein (apo) E2(Cys-158), an apoE variant associated with the human genetic disorder type III hyperlipoproteinemia (HLP). Male transgenic rabbits fed normal chow had up to 8-fold (289 +/- 148 mg/dl) and 15-fold (697 +/- 452 mg/dl) increases in plasma total cholesterol and triglycerides, respectively, compared with nontransgenic males. Female transgenic rabbits had only a modest hyperlipidemia (total cholesterol, 140 +/- 46 mg/dl; total triglycerides, 174 +/- 66 mg/dl). Both sexes displayed the hallmarks fo type III HLP: beta-migrating very low density lipoproteins (beta-VLDL) (intestinal and hepatic remnant lipoproteins) and significantly increased VLDL and intermediate density lipoproteins. Apolipoprotein E2-containing VLDL particles were cleared from teh circulation more slowly and were more resistant to lipoprotein lipase-mediated lipolysis than normal VLDL. Only females had increased high density lipoproteins (HDL) (40%), which were shifted from typical small HDL to larger HDL1. Plasma apoE2 was predominantly associated with beta-VLDL in males and with HDL in females. To ascertain reasons for the phenotypic gender difference, we treated male transgenic rabbits with 17alpha-ethinyl estradiol. Estrogen treatment for 10 days dramatically decreased total cholesterol (73%) and triglycerides (89%) and converted beta-VLDL to pre-beta-migrating VLDL. Concomitantly, lipoprotein lipase and hepatic lipase activities increased by 90%, low density lipoprotein receptor activity was stimulated significantly, apoE2 was redistributed to HDL, and HDL were converted to HDL1. Conversely, ovariectomy in female transgenic rabbits significantly increased total cholesterol (75%), triglycerides (117%), and beta-VLDL, while decreasing lipoprotein lipase and hepatic lipase activities by 35% and redistributing apoE2 to the beta-VLDL. Thus, estrogen status appears to be responsible for much of the gender difference of the lipoprotein phenotype, mainly by modulating both lipase and low density lipoprotein receptor activities. Furthermore, transgenic rabbits fed normal chow for 11 months developed fatty streaks, and some had more advanced atherosclerotic lesions, especially around the aortic arch and proximal abdominal aorta. The lesions were more extensive in males, roughly correlating with the magnitude of the hyperlipidemia. Therefore, high plasma levels of human apoE2 in transgenic rabbits result in a type III HLP phenotype, in which males have both more severe hyperlipidemia and more extensive atherosclerosis than females.


Subject(s)
Apolipoproteins E/genetics , Arteriosclerosis/drug therapy , Estrogens/therapeutic use , Hyperlipoproteinemias/drug therapy , Animals , Animals, Genetically Modified , Apolipoprotein E2 , Apolipoproteins E/blood , Arteriosclerosis/complications , Female , Hyperlipoproteinemias/blood , Hyperlipoproteinemias/complications , Lipids/blood , Lipolysis , Lipoproteins, VLDL/blood , Male , Phenotype , Rabbits
10.
J Clin Invest ; 100(1): 180-8, 1997 Jul 01.
Article in English | MEDLINE | ID: mdl-9202070

ABSTRACT

All classes of lipoproteins considered to be atherogenic contain apo-B100 or apo-B48. However, there is a distinct paucity of data regarding whether lipoproteins containing apo-B48 or apo-B100 differ in their intrinsic ability to promote the development of atherosclerosis. To address this issue, we compared the extent of atherosclerosis in three groups of animals: apo-E-deficient mice (apo-B+/+apo-E-/-) and apo-E-deficient mice that synthesize exclusively either apo-B48 (apo-B48/48apo-E-/-) or apo-B100 (apo-B100/100apo-E-/-). Mice (n = 25 in each group) were fed a chow diet for 200 days, and plasma lipid levels were assessed throughout the study. Compared with the levels in apo-B+/+apo-E-/- mice, the total plasma cholesterol levels were higher in the apo-B48/48apo-E-/- mice and were lower in the apo-B100/100apo-E-/- mice. However, the ranges of cholesterol levels in the three groups overlapped. Compared with those in the apo-B+/+apo-E-/- mice, atherosclerotic lesions were more extensive in the apo-B48/48apo-E-/- mice and less extensive in the apo-B100/100apo-E-/- mice. Once again, however, there was overlap among the three groups. The extent of atherosclerosis in each group of mice correlated significantly with plasma cholesterol levels. In mice from different groups that had similar cholesterol levels, the extent of atherosclerosis was quite similar. Thus, susceptibility to atherosclerosis was dependent on total cholesterol levels. Whether mice synthesized apo-B48 or apo-B100 did not appear to have an independent effect on susceptibility to atherosclerosis.


Subject(s)
Apolipoproteins B/biosynthesis , Apolipoproteins E/deficiency , Arteriosclerosis/genetics , Animals , Aorta/pathology , Apolipoprotein B-100 , Apolipoprotein B-48 , Apolipoproteins B/blood , Arteriosclerosis/blood , Arteriosclerosis/pathology , Cholesterol/blood , Disease Susceptibility , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Mice, Mutant Strains , Muscle, Smooth, Vascular/pathology
11.
J Lipid Res ; 38(5): 1002-13, 1997 May.
Article in English | MEDLINE | ID: mdl-9186917

ABSTRACT

The cellular location of hepatic lipase was investigated in transgenic rabbits that expressed human hepatic lipase in the liver. The binding of monoclonal antibodies to human hepatic lipase, as detected by either fluorescence-tagged or gold-conjugated secondary antibodies, showed that hepatic lipase was concentrated at the surfaces of hepatic sinusoids. This distribution was the same as observed in the human liver. At the ultrastructural level, immunogold labeling of the space of Disse showed hepatic lipase on both lumenal and sublumenal surfaces of rabbit liver sinusoidal endothelial cells. An equivalent amount of hepatic lipase also was found on the external surfaces of hepatocyte microvilli in the space of Disse, as well as in the interhepatocyte spaces. The distribution suggests that a majority of the hepatic lipase produced by the liver is associated with hepatocyte surfaces, consistent with the functions of this enzyme in lipoprotein metabolism.


Subject(s)
Endothelium/enzymology , Lipase/metabolism , Liver/enzymology , Animals , Cell Membrane/enzymology , Endothelium/ultrastructure , Humans , Immunohistochemistry , Liver/cytology , Microscopy, Electron , Rabbits
12.
Infect Immun ; 64(8): 3210-7, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8757855

ABSTRACT

Group B Neisseria meningitidis causes systemic disease, including meningitis, after initial colonization and subsequent penetration of nasopharyngeal mucosa, a tissue which is richly populated by macrophages. In an initial effort to characterize the interaction of N. meningitidis and mature human macrophages, the influence of the alpha2-->8) -linked polysialic acid capsule on the interaction of N. meningitidis with human monocyte-derived macrophages was investigated with a capsulate case isolate and an isogenic Tn916-derived noncapsulate transformant. The capsulate strain was fourfold less adherent to the macrophage surface after cold incubation, although adherence of both strains was significantly increased after opsonization with nonimmune C5-depleted serum. When opsonized inocula were adjusted so that they adhered to macrophages in equal numbers, the two strains were internalized at equivalent rates and both entered membrane-bound compartments (phagosomes). Colocalization of bacteria with the late endosomal and lysosomal marker lysosome-associated membrane protein revealed that fusion of lysosomes with phagosomes containing the capsulate organism was significantly reduced 10 and 30 min after entry, but by 1 h, no difference between the strains was observed. Once internalized, meningococci were effectively killed, although more rapid killing of the capsulate strain was observed over the first 3 h. These results indicate that the (alpha2-->8)-linked polysialic acid capsule modifies the interaction of meningococci with human macrophages at multiple steps, including adherence to the macrophage surface and phagosome-lysosome fusion. Moreover, the discordance between the kinetics of phagosome- lysosome fusion and bacterial killing suggests that a nonlysosomal mechanism may be responsible for a significant fraction of macrophage killing of N. meningitidis.


Subject(s)
Bacterial Capsules/metabolism , Macrophages/microbiology , Neisseria meningitidis/physiology , Sialic Acids/metabolism , Antigens, CD/metabolism , Bacterial Adhesion , Bacterial Capsules/genetics , Humans , Lysosomal Membrane Proteins , Lysosomes , Membrane Glycoproteins/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Mutagenesis, Insertional , Neisseria meningitidis/classification , Phagocytosis , Phagosomes , Sialic Acids/genetics
13.
J Biol Chem ; 271(12): 7004-11, 1996 Mar 22.
Article in English | MEDLINE | ID: mdl-8636130

ABSTRACT

Previously, we showed that a subpopulation of the major platelet integrin, alphaIIbbeta3, co-sediments from detergent lysates with talin and other membrane skeleton proteins. Once alphaIIbbeta3 has bound adhesive ligand in a platelet aggregate, the detergent-insoluble alphaIIbbeta3 redistributes (along with the detergent-insoluble membrane skeleton proteins and a variety of signaling molecules) to a fraction that contains cytoplasmic actin filaments. Concomitantly, certain signaling molecules are activated. The present study shows that, in intact platelets, alphaIIbbeta3 forms clusters when occupied by ligand and is selectively moved into the open canalicular system; alphaIIbbeta3 that has not bound ligand remains diffusely distributed at the periphery of the cell. When cytoplasmic actin filaments are depolymerized by cytochalasins, the ability of alphaIIbbeta3 to bind ligand is decreased, and the movement of ligand-occupied alphaIIbbeta3 is prevented. Together with the previous findings, these results suggest that (i) membrane skeleton-associated alphaIIbbeta3 is selectively induced to bind ligand in activated platelets, (ii) ligand-induced transmembrane signaling causes an altered association of membrane skeleton-associated alphaIIbbeta3 with the cytoplasmic component of the cytoskeleton, (iii) ligand-induced cytoskeletal reorganizations stabilize the interaction between ligand and integrin, and (iv) ligand-occupancy triggers cytoskeletal reorganizations that result in selective movements of occupied ligand.


Subject(s)
Blood Platelets/metabolism , Cytoskeleton/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Actins/metabolism , Adult , Cytochalasin B/pharmacology , Fibronectins/metabolism , Humans , Ligands , Microscopy, Confocal , Microscopy, Electron , Microscopy, Fluorescence , Protein Binding
14.
J Cell Biol ; 132(1-2): 49-61, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8567729

ABSTRACT

Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms. Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+]i). Indeed, increases in [Ca2+]i are essential for phagosome-granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes. Since several intracellular pathogens survive in macrophage phagosomes that do not fuse with lysosomes, we examined the regulation of phagosome-lysosome fusion in macrophages. Macrophages (M phi) were treated with 12.5 microM bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N',-tetraacetic acid tetraacetoxymethyl ester (MAPT/AM), a cell-permeant calcium chelator which reduced resting cytoplasmic [Ca2+]; from 80 nM to < or = 20 nM and completely blocked increases in [Ca2+]i in response to multiple stimuli, even in the presence of extracellular calcium. Subsequently, M phi phagocytosed serum-opsonized zymosan, staphylococci, or Mycobacterium bovis. Microbes were enumerated by 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LAMP-1) as a membrane marker and rhodamine dextran as a content marker for lysosomes. Confirmation of phagosome-lysosome fusion by electron microscopy validated the fluorescence microscopy findings. We found that phagosome-lysosome fusion in M phi occurs noramlly at very low [Ca2+]i (< or = 20 nM). Kinetic analysis showed that in M phi none of the steps leading from particle binding to eventual phagosome-lysosome fusion are regulated by [Ca2+]i in a rate-limiting way. Furthermore, confocal microscopy revealed no difference in the intensity of LAMP-1 immunofluorescence in phagolysosome membranes in calcium-buffered vs. control macrophages. We conclude that neither membrane recognition nor fusion events in the phagosomal pathway in macrophages are dependent on or regulated by calcium.


Subject(s)
Calcium/metabolism , Lysosomes/physiology , Macrophages/physiology , Membrane Fusion , Phagocytosis/physiology , Phagosomes/physiology , Animals , Antigens, CD/isolation & purification , Cell Line , Chelating Agents/pharmacology , Dextrans , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Fluorescent Antibody Technique , Humans , Lysosomal Membrane Proteins , Membrane Glycoproteins/isolation & purification , Mice , Microscopy, Immunoelectron , Mycobacterium , Rhodamines , Staphylococcus , Zymosan
15.
J Clin Invest ; 96(5): 2170-9, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7593602

ABSTRACT

apoE deficiency causes hyperlipidemia and premature atherosclerosis. To determine if macrophage-specific expression of apoE would decrease the extent of atherosclerosis, we expressed human apoE in macrophages of apoE-null mice (apoE-/-) and assessed the effect on lipid accumulation in cells of the arterial wall. Macrophage-specific expression of human apoE in normal mice was obtained by use of the visna virus LTR. These animals were bred with apoE-/- mice to produce animals hemizygous for expression of human apoE in macrophages in the absence of murine apoE (apoE-/-,hTgE+/0). Low levels of human apoE mRNA were present in liver and spleen and high levels in lung and peritoneal macrophages. Human apoE was secreted by peritoneal macrophages and was detected in Kupffer cells of the liver. Human apoE in the plasma of apoE-/-,hTgE+/0 mice (n = 30) was inversely correlated (P < 0.005) with the plasma cholesterol concentration. After 15 wk on a normal chow diet, atherosclerosis was assessed in apoE-/-,hTgE+/0 animals and in apoE-/-,hTgE0/0 littermates matched for plasma cholesterol level (approximately 450 mg/dl) and lipoprotein profile. There was significantly less atherosclerosis in both the aortic sinus and in the proximal aorta (P < 0.0001) in the animals expressing the human apoE transgene. In apo-E-/-,hTgE+/0 animals, which had detectable atherosclerotic lesions, human apoE was detected in the secretory apparatus of macrophage-derived foam cells in the arterial wall. The data demonstrate that expression of apoE by macrophages is antiatherogenic even in the presence of high levels of atherogenic lipoproteins. The data suggest that apoE prevents atherosclerosis by promoting cholesterol efflux from cells of the arterial wall.


Subject(s)
Apolipoproteins E/biosynthesis , Arteriosclerosis/metabolism , Hypercholesterolemia/metabolism , Macrophages/metabolism , Animals , Apolipoproteins E/genetics , Arteriosclerosis/genetics , Arteriosclerosis/pathology , Cells, Cultured , Cholesterol/blood , Female , Foam Cells/metabolism , Gene Expression , Gene Transfer Techniques , Humans , Hypercholesterolemia/genetics , Hypercholesterolemia/pathology , Male , Mice , Mice, Transgenic
16.
Arterioscler Thromb Vasc Biol ; 15(11): 1911-6, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7583571

ABSTRACT

Transgenic mice expressing transgenes for both human apolipoprotein B-100 (h-apoB) and apolipoprotein(a) [apo(a)] were fed a high-fat, atherogenic diet for 14 weeks to examine the effect of lipoprotein(a) [Lp(a)]on the development of aortic fatty lesions. The extent of lesions in the proximal region of the aorta of Lp(a) mice was measured by use of a computer-assisted image analysis of 20 sections per animal and compared with that of nontransgenic mice as well as mice expressing either the apo(a) or h-apoB transgene. The control (n = 23) and apo(a) (n = 22) transgenic mice had very small mean lesions areas (607 versus 128 microns2 per section). The h-apoB-expressing mice (n = 20) had significantly higher mean lesion areas (3288 microns2 per section) than either the control or apo(a) transgenic animals. Coexpression of apo(a) and h-apoB transgenes resulted in only a modest increase in lesion area (4678 microns2 per section, n = 19). Thus, the expression of human apo(a) in C57BL/6/SJL hybrid mice fed an atherogenic diet failed to significantly potentiate the development of aortic fatty lesions in the absence or presence of high levels of h-apoB.


Subject(s)
Apolipoproteins B/biosynthesis , Apolipoproteins/biosynthesis , Arteriosclerosis/metabolism , Lipoprotein(a) , Animals , Aorta/pathology , Apolipoproteins/genetics , Apolipoproteins B/genetics , Apoprotein(a) , Arteriosclerosis/etiology , Arteriosclerosis/pathology , Diet, Atherogenic , Gene Transfer Techniques , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic
17.
J Clin Invest ; 96(4): 1987-2001, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7560092

ABSTRACT

The mechanism by which pleural mesothelial cells, the likely progenitor cells of asbestos-induced mesothelioma, recognize and internalize crocidolite asbestos is unknown. Because incubation of asbestos fibers with serum increases their association with cells, we asked whether a protein coat on asbestos increased internalization of fibers via specific cellular receptors. Coating crocidolite with citronectin, but not with fibronectin or other proteins, increased fiber internalization by rabbit pleural mesothelial cells, as measured by a new technique using fluorescence confocal microscopy. Receptors for vitronectin, alpha v beta 3 and alpha v beta 5, were identified on mesothelial cells. Inhibiting vitronectin receptors by plating cells on a vitronectin substrate or incubating cells with excess soluble vitronectin reduced internalization of vitronectin-coated crocidolite. Inhibition of alpha v beta 5, but not alpha v beta 3, with blocking antibodies similarly reduced internalization. In addition, alpha v beta 5, but not alpha v beta 3, showed immunocytochemical colocalization with fibers. Of biologic relevance, coating crocidolite with serum also increased internalization via alpha v beta 5, an effect dependent on the vitronectin in serum. We conclude that pleural mesothelial cells recognize and internalize vitronectin- and serum-coated asbestos via the integrin alpha v beta 5. Since integrins initiate some of the same signaling pathways as does asbestos, our findings may provide insights into the mechanisms of asbestos-induced biologic effects.


Subject(s)
Asbestos, Crocidolite/metabolism , Integrins/physiology , Pleura/metabolism , Vitronectin/pharmacology , Amino Acid Sequence , Animals , Cells, Cultured , Epithelium/metabolism , Fluorescence , Molecular Sequence Data , Oligopeptides/pharmacology , Rabbits , Receptors, Vitronectin/physiology
18.
J Biol Chem ; 270(27): 16302-7, 1995 Jul 07.
Article in English | MEDLINE | ID: mdl-7608197

ABSTRACT

The glycoprotein (GP) Ib-IX-V complex comprises four polypeptides: the subunits of the GP Ib-IX complex (GP Ib alpha, GP Ib beta, GP IX) and GP V. To determine the requirements for cell-surface expression of GPV, we transiently expressed the recombinant polypeptide in wild-type Chinese hamster ovary (CHO) cells by cotransfection with plasmids for the subunits of the GP Ib-IX complex and in CHO cells that stably express different combinations of the GP Ib-IX complex subunits. Glycoprotein V expressed alone was detectable on the cell surface, and the level was not augmented by cotransfection with any one of the subunits of the GP Ib-IX complex. However, when GP V was expressed in cells that stably express combinations of GP Ib-IX complex subunits, its expression on the cell surface was greater in all the cell lines that contained GP Ib alpha than in wild-type CHO cells. That GP V associates with GP Ib alpha was also suggested by confocal microscopy studies: GP V colocalized with GP Ib alpha in CHO alpha beta IX (cells that express GP Ib alpha, GP Ib beta, and GP IX), CHO alpha beta, and CHO alpha IX cells, but did not colocalize with GP Ib beta in CHO beta IX cells. Similarly, immunoprecipitation of GP V from cells expressing GP Ib alpha led to coprecipitation of the latter polypeptide; neither GP Ib beta nor GP IX coprecipitated with GP V from CHO beta IX cells. Taken together, these data indicate that GP V associates with the GP Ib-IX complex through a direct interaction with GP Ib alpha and establish the topology of the GP Ib-IX-V subunits on the cell surface.


Subject(s)
Cell Membrane/metabolism , Platelet Membrane Glycoproteins/metabolism , Animals , CHO Cells , Cell Membrane/ultrastructure , Cricetinae , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Regulation , Macromolecular Substances , Microscopy, Confocal , Models, Molecular , Platelet Membrane Glycoproteins/biosynthesis , Platelet Membrane Glycoproteins/genetics , Precipitin Tests , Protein Binding , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Transfection
19.
J Clin Invest ; 95(5): 2246-57, 1995 May.
Article in English | MEDLINE | ID: mdl-7738190

ABSTRACT

We previously generated transgenic mice expressing human apolipoprotein (apo-) B and demonstrated that the plasma of chow-fed transgenic animals contained markedly increased amounts of LDL (Linton, M. F., R. V. Farese, Jr., G. Chiesa, D. S. Grass, P. Chin, R. E. Hammer, H. H. Hobbs, and S. G. Young 1992. J. Clin. Invest. 92:3029-3037). In this study, we fed groups of transgenic and nontransgenic mice either a chow diet or a diet high in fat (16%) and cholesterol (1.25%). Lipid and lipoprotein levels were assessed, and after 18 wk of diet, the extent of aortic atherosclerotic lesions in each group of animals was quantified. Compared with the female transgenic mice on the chow diet, female transgenic mice on the high-fat diet had higher plasma levels of cholesterol (312 +/- 17 vs 144 +/- 7 mg/dl; P < 0.0001) and human apo-B (120 +/- 8 vs 84 +/- 3 mg/dl; P < 0.0001). The higher human apo-B levels were due to increased plasma levels of human apo-B48; the human apo-B100 levels did not differ in animals on the two diets. In mice on the high-fat diet, most of the human apo-B48 and apo-B100 was found in LDL-sized particles. Compared with nontransgenic mice on the high-fat diet, the transgenic animals on the high-fat diet had significantly increased levels of total cholesterol (312 +/- 17 vs 230 +/- 19 mg/dl; P < 0.0001) and non-HDL cholesterol (283 +/- 17 vs 193 +/- 19 mg/dl; P < 0.0001). The extent of atherosclerotic lesion development within the ascending aorta was quantified by measuring total lesion area in 60 progressive sections, using computer-assisted image analysis. Neither the chow-fed transgenic mice nor the chow-fed nontransgenic mice had significant atherosclerotic lesions. Nontransgenic animals on the high-fat diet had relatively small atherosclerotic lesions (< 15,000 microns 2/section), almost all of which were confined to the proximal 400 microns of the aorta near the aortic valve. In contrast, transgenic animals on the high-fat diet had extensive atherosclerotic lesions (> 160,000 microns 2/section) that were widely distributed throughout the proximal 1,200 microns of the aorta. Thus, human apo-B expression, in the setting of a diet rich in fats, causes severe atherosclerosis in mice.


Subject(s)
Apolipoproteins B/biosynthesis , Arteriosclerosis/physiopathology , Diet, Atherogenic , Dietary Fats , Animals , Aorta, Thoracic/pathology , Aorta, Thoracic/ultrastructure , Apolipoprotein B-100 , Apolipoproteins B/blood , Apolipoproteins B/genetics , Arteriosclerosis/genetics , Arteriosclerosis/pathology , Base Sequence , Cholesterol/blood , Cholesterol, HDL/blood , Crosses, Genetic , Female , Humans , Intestinal Mucosa/metabolism , Kidney/metabolism , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Transgenic , Microscopy, Electron , Molecular Sequence Data , Oligodeoxyribonucleotides , Organ Specificity , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Sex Characteristics , Sex Factors , Triglycerides/blood
20.
J Lipid Res ; 36(3): 583-92, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7539827

ABSTRACT

Heparan sulfate proteoglycans (HSPG) are involved in the binding and uptake of apolipoprotein (apo) E-enriched remnant lipoproteins by cultured cells in vitro. To define the role of hepatic HSPG in remnant lipoprotein clearance in vivo, heparinase (30 units) was infused intravenously into mice to hydrolyze the liver HSPG and determine the effect of HSPG hydrolysis on remnant clearance by the liver. Liver HSPG were prelabeled by peritoneal injection of [35S]Na2SO4. Injection of heparinase decreased the amount of 35S-labeled liver HSPG by approximately 20-40% within 10-15 min. Heparinase infusion significantly inhibited the clearance of chylomicrons, chylomicron remnants, chylomicron remnants + apoE, rabbit beta-very low density lipoproteins (beta-VLDL), and beta-VLDL + apoE. Compared with saline injection in control mice, heparinase injection retarded the plasma clearance of the remnants by 1.5- to 2-fold and decreased liver uptake by 1.3- to 1.6-fold. Confocal fluorescence microscopy of thick slices of liver from mice injected with 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine-labeled beta-VLDL + apoE revealed markedly less intense fluorescence from hepatocytes in heparinase-treated animals compared with those in saline-treated control animals. Intravenous heparinase infusion did not inhibit the clearance of mouse low density lipoproteins (LDL), a ligand for the LDL receptor, and did not affect the clearance of alpha 2-macroglobulin, a ligand for the LDL receptor-related protein. The results suggest an important role of the liver HSPG in remnant clearance in vivo.


Subject(s)
Heparitin Sulfate/metabolism , Lipoproteins/blood , Lipoproteins/metabolism , Liver/metabolism , Polysaccharide-Lyases/administration & dosage , Proteoglycans/metabolism , Animals , Apolipoproteins E/blood , Apolipoproteins E/metabolism , Dogs , Heparan Sulfate Proteoglycans , Heparin Lyase , In Vitro Techniques , Infusions, Intravenous , Lactoferrin/metabolism , Lipoproteins, VLDL/blood , Lipoproteins, VLDL/metabolism , Liver/drug effects , Low Density Lipoprotein Receptor-Related Protein-1 , Mice , Rabbits , Receptors, Immunologic/metabolism , Sulfates/metabolism , alpha-Macroglobulins/metabolism
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