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1.
Thromb Haemost ; 106(5): 814-9, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21946866

ABSTRACT

Cells of both the innate and adaptive immune system participate in the development of atherosclerosis, a chronic inflammatory disorder of medium and large arteries. Natural killer T (NKT) cells express surface markers characteristic of natural killer cells and conventional T cells and bridge the innate and adaptive immune systems. The development and activation of NKT cells is dependent upon CD1d, a MHC-class I-type molecule that presents lipids, especially glycolipids to the T cell receptors on NKT cells. There are two classes of NKT cells; invariant NKT cells that express a semi-invariant T cell receptor and variant NKT cells. This review summarises studies in murine models in which the effect of the activation, overexpression or deletion of NKT cells or only invariant NKT cells on atherosclerosis has been examined.


Subject(s)
Arteries/immunology , Atherosclerosis/immunology , Inflammation/immunology , Lipoproteins/metabolism , Natural Killer T-Cells/immunology , Adaptive Immunity , Animals , Antigens, CD1d/metabolism , Arteries/metabolism , Atherosclerosis/metabolism , Humans , Immunity, Innate , Inflammation/metabolism , Natural Killer T-Cells/metabolism , Phenotype
2.
Curr Pharm Des ; 16(28): 3173-84, 2010.
Article in English | MEDLINE | ID: mdl-20687877

ABSTRACT

Elevations of HDL levels or modifying the inflammatory properties of HDL are being evaluated as possible treatment of atherosclerosis, the underlying mechanism responsible for most cardiovascular diseases. A promising approach is the use of small HDL apoprotein-related mimetic peptides. A number of peptides mimicking the repeating amphipathic α-helical structure in apoA-I, the major apoprotein in HDL, have been examined in vitro and in animal models. Several peptides have been shown to reduce early atherosclerotic lesions, but not more mature lesions unless coadministered with statins. These peptides also influence the vascular biology of the vessel wall and protect against other acute and chronic inflammatory diseases. The biologically active peptides are capable of reducing the pro-inflammatory properties of LDL and HDL, likely due to their high affinity for oxidized lipids. They are also capable of influencing other processes, including ABCA1 mediated activation of JAK-2 in macrophages, which may contribute to their anti-atherogenic function. The initial studies involved monomeric 18 amino acid peptides, but tandem peptides are being investigated for their anti-atherogenic and anti-inflammatory properties as they more closely resemble the repeating structure of apoA-I. Peptides based on other HDL associated proteins such as apoE, apoJ and SAA have also been studied. Their mechanism of action appears to be distinct from the apoA-I based mimetics.


Subject(s)
Apolipoproteins/physiology , Atherosclerosis/drug therapy , Inflammation/drug therapy , Lipoproteins, HDL/drug effects , Peptides/therapeutic use , Animals , Anti-Inflammatory Agents/therapeutic use , Apolipoproteins/chemistry , Atherosclerosis/physiopathology , Cholesterol/metabolism , Clusterin/chemistry , Clusterin/physiology , Humans , Inflammation/physiopathology , Molecular Mimicry , Peptides/chemistry , Peptides/pharmacology , Protein Structure, Secondary
3.
Curr Drug Targets ; 8(11): 1203-14, 2007 Nov.
Article in English | MEDLINE | ID: mdl-18045099

ABSTRACT

Atherosclerosis is a chronic inflammatory reaction that is initiated in response to hyperlipidemia and the retention and modification of lipids within the vascular wall. Chronic inflammatory states lead to steady low-level induction of the acute phase reaction and chronic inflammation is associated with elevated cardiovascular disease and atherosclerosis. The acute phase reaction is mediated by cytokines and results in significant changes in the plasma level of several proteins referred to as acute phase proteins. The liver is a major source of these proteins. Several recent studies in humans have shown that levels of acute phase proteins are modified in patients with established cardiovascular disease or are predictors of future disease. Whether these acute phase proteins are a biomarker of inflammation or have a direct role in the development of atherosclerosis is not clear. Murine models of atherosclerosis have been used to address the role of acute phase proteins in atherosclerosis. Modification of the expression level of these proteins has shown that the individual acute phase proteins are either pro-atherogenic or anti-atherogenic. The absence of an overall trend is perhaps not surprising given the complex nature of the acute phase response.


Subject(s)
Acute-Phase Proteins/physiology , Atherosclerosis/blood , Atherosclerosis/etiology , Acute-Phase Proteins/biosynthesis , Acute-Phase Proteins/genetics , Animals , Atherosclerosis/genetics , Atherosclerosis/pathology , Biomarkers/blood , Chronic Disease , Humans , Mice
4.
Biochemistry ; 40(45): 13670-80, 2001 Nov 13.
Article in English | MEDLINE | ID: mdl-11695916

ABSTRACT

We have used adenovirus-mediated gene transfer in apoA-I-deficient (A-I-/-) mice to probe the in vivo assembly and metabolism of HDL using apoA-I variants, focusing primarily on the role of the C-terminal 32 amino acids (helices 9-10). Lipid, lipoprotein, and apoA-I analyses showed that plasma levels of apoA-I and HDL of the mutants were 40-88% lower than that of wild type (WT) human apoA-I despite comparable levels of expression in the liver. WT apoA-I and mutant 1 (P165A, E172A) formed spherical particles with the size and density of HDL2 and HDL3. Mutant 2 (E234A, E235A, K238A, K239A) generated spherical particles with density between HDL2 and HDL3. Mutant 3 (L211V, L214V, L218V, L219V) and mutant 4 (L222K, F225K, F229K), which have substitutions of hydrophobic residues in the C-terminus, generated discoidal HDL particles indicating a defect in their conversion to mature spherical HDL. Significant amounts of mutant 4 and mutant 5 (truncated at residue 219) were found in the lipid poor fractions after ultracentrifugation of the plasma (18 and 35%, respectively, of total apoA-I). These findings suggest that hydrophobic residues in and/or between helices 9 and 10 are important for the maturation of HDL in vivo.


Subject(s)
Apolipoprotein A-I/metabolism , Lipoproteins, HDL/metabolism , Liver/metabolism , Adenoviridae/genetics , Animals , Apolipoprotein A-I/blood , Apolipoprotein A-I/deficiency , Apolipoprotein A-I/genetics , Cholesterol, HDL/blood , Dimyristoylphosphatidylcholine/metabolism , Gene Deletion , Gene Transfer Techniques , Genetic Vectors , Humans , Lipids/blood , Lipoproteins, HDL/blood , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Phosphatidylcholine-Sterol O-Acyltransferase/metabolism , Protein Structure, Secondary , RNA, Messenger/metabolism
5.
Neurochem Int ; 39(5-6): 427-34, 2001.
Article in English | MEDLINE | ID: mdl-11578778

ABSTRACT

Large numbers of activated glia are a common pathological feature of many neurodegenerative disorders, including Alzheimer's disease (AD). Several different stimuli, including lipopolysaccharide (LPS), dibutyryl (db)cAMP, and aged amyloid-beta 1-42 (A beta), can induce glial activation in vitro, as measured by morphological changes and the production of pro-inflammatory cytokines and oxidative stress molecules. Only A beta-induced activation is attenuated by the addition of exogenous apolipoprotein E (apoE)-containing particles. In addition, only A beta also induces an increase in the amount of endogenous apoE, the primary apolipoprotein expressed by astrocytes in the brain. The functional significance of the increase in apoE appears to be to limit the inflammatory response. Indeed, compared to wild type mice, glial cells cultured from apoE knockout mice exhibit an enhanced production of several pro-inflammatory markers in response to treatment with A beta and other activating stimuli. The mechanism for both the A beta-induced glial activation and the increase in apoE appears to involve apoE receptors, a variety of which are expressed by both neurons and glia. Experiments using receptor associated protein (RAP), an inhibitor of apoE receptors with a differential affinity for the low-density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), revealed that LRP mediates A beta-induced glial activation, while LDLR mediates the A beta-induced changes in apoE levels. In summary, both an apoE receptor agonist (apoE) and an antagonist (RAP) inhibit A beta-induced glial cell activation. Thus, apoE receptors appear to translate the presence of extracellular A beta into cellular responses, both initiating glial cell activation and limiting its scope by inducing apoE, an anti-inflammatory agent.


Subject(s)
Amyloid beta-Peptides/physiology , Apolipoproteins E/physiology , Encephalitis/physiopathology , Low Density Lipoprotein Receptor-Related Protein-1/physiology , Neuroglia/physiology , Animals , Humans
6.
Arterioscler Thromb Vasc Biol ; 21(6): 1011-6, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11397712

ABSTRACT

To determine whether T cells and B cells influence lipid metabolism and atherosclerosis, we crossed apolipoprotein E-deficient (apoE degrees ) mice with recombination activating gene 2-deficient (RAG2 degrees ) mice. Total plasma cholesterol levels were approximately 20% higher in male apoE degrees mice compared with the apoE degrees RAG2 degrees mice at 8 weeks of age, and plasma triglyceride levels were 2.5-fold higher in the apoE degrees mice even when plasma cholesterol levels were similar. Male mice with plasma cholesterol levels between 400 and 600 mg/dL at 8 weeks of age were euthanized at 27 and 40 weeks of age. The aortic root lesion area in the apoE degrees RAG2 degrees mice, compared with that in the immune-competent apoE degrees mice, was 81% and 57% smaller at 27 and 40 weeks of age, respectively. In contrast, there was no difference in the size of the brachiocephalic trunk lesions. Similar results were obtained with mice euthanized at 40 weeks of age that had 8-week cholesterol levels between 300 and 399 mg/dL. In apoE degrees RAG2 degrees mice, aortic root atherosclerosis was more profoundly suppressed at lower cholesterol levels. Thus, T and B cells and their products differentially influence the development of atherosclerosis at different sites. We also demonstrate a profound effect of the immune system on plasma lipid homeostasis.


Subject(s)
Apolipoproteins E/genetics , Arteriosclerosis/immunology , Immunocompromised Host , Lipoproteins/blood , Animals , Aorta/pathology , Arteriosclerosis/blood , Arteriosclerosis/pathology , Brachiocephalic Trunk/pathology , DNA-Binding Proteins/genetics , Lipids/blood , Male , Mice , Mice, Inbred C57BL , Mice, Knockout
7.
Curr Opin Lipidol ; 12(2): 167-73, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11264988

ABSTRACT

Atherosclerosis bears many features of a chronic inflammation that affects the intima of large and medium-sized arteries. In recent years apolipoprotein E-deficient and LDL receptor-deficient mice have been used to examine the effects of various gene products on the development of atherosclerosis. In the present review the effects of genetics, apolipoprotein E, inflammatory gene modifiers, lipoprotein modifications, lipoprotein receptors, vessel wall expression of lipoprotein-metabolizing enzymes, and the atheroprotective role of HDL on atherosclerosis in these mice are discussed. The importance of examining lesions that are more advanced than fatty streaks and careful histologic and immunologic examination of lesion composition is emphasized.


Subject(s)
Arteriosclerosis , Disease Models, Animal , Animals , Apolipoproteins E/physiology , Arteriosclerosis/etiology , Arteriosclerosis/genetics , Arteriosclerosis/prevention & control , Genetic Predisposition to Disease , Inflammation/genetics , Lipoproteins/metabolism , Lipoproteins, HDL/physiology , Mice , Receptors, Lipoprotein/physiology
8.
Biochemistry ; 39(51): 16119-24, 2000 Dec 26.
Article in English | MEDLINE | ID: mdl-11123940

ABSTRACT

Extracellular senile plaques composed predominantly of fibrillar amyloid-beta (Abeta) are a major neuropathological feature of Alzheimer's disease (AD). Genetic evidence and in vivo studies suggest that apolipoprotein E (apoE) may contribute to amyloid clearance and/or deposition. In vitro studies demonstrate that native apoE2 and E3 form an SDS-stable complex with Abeta(1-40), while apoE4 forms little such complex. Our current work extends these observations by presenting evidence that apoE3 also binds to Abeta(1-42) and with less avidity to modified species of the peptide found in senile plaque cores. These modified peptides include a form that originates at residue 3-Glu as pyroglutamyl and another with isomerization at the 1-Asp and 7-Asp positions. In addition, we used binding reactions between apoE3 and various Abeta fragments, as well as binding reactions with apoE3 and Abeta(1-40) plus Abeta fragments as competitors, to identify the domain(s) of Abeta involved in the formation of an SDS-stable complex with apoE3. Residues 13-28 of Abeta appear to be necessary, while complex formation is further enhanced by the presence of residues at the C-terminus of the peptide. These results contribute to our understanding of the biochemical basis for the SDS-stable apoE3/Abeta complex and support the hypothesis that Abeta can be transported in vivo complexed with apoE. This complex may then be cleared from the interstitial space by apoE receptors in the brain or become part of an extracellular amyloid deposit.


Subject(s)
Amyloid beta-Peptides/metabolism , Apolipoproteins E/metabolism , Peptide Fragments/metabolism , Amino Acid Substitution , Amyloid beta-Peptides/pharmacology , Apolipoprotein E3 , Apolipoproteins E/pharmacology , Binding, Competitive , Cell Line , Electrophoresis, Polyacrylamide Gel , Humans , Macromolecular Substances , Peptide Fragments/pharmacology , Protein Structure, Tertiary , Sodium Dodecyl Sulfate
9.
J Biol Chem ; 275(43): 33974-80, 2000 Oct 27.
Article in English | MEDLINE | ID: mdl-10940295

ABSTRACT

We have previously shown that beta-amyloid (Abeta) induces astrocyte activation in vitro and that this reaction is attenuated by the addition of exogenous apolipoprotein E (apoE)-containing particles. However, the effects of Abeta on endogenous apoE and apoJ levels and the potential role of apoE receptors in astrocyte activation have not been addressed. Three activating stimuli (lipopolysaccharide, dibutyryl cAMP, and aged Abeta 1-42) were used to induce activation of rat astrocyte cultures, as assessed by changes in morphology and an increase in interleukin-1beta. However, only Abeta also induced approximately 50% reduction in the amount of released apoE and apoJ and an 8-fold increase in the levels of cell-associated apoE and apoJ. Experiments using two concentrations of receptor-associated protein, an inhibitor of apoE receptors with a differential affinity for the low density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), suggest that LRP mediates Abeta-induced astrocyte activation, whereas LDLR mediates the Abeta-induced changes in apoE levels. Receptor-associated protein had no effect on apoJ levels or on activation by either dibutyryl cAMP or lipopolysaccharide. These data suggest that apoE receptors translate the presence of extracellular Abeta into cellular responses, both initiating and modulating the inflammatory response induced by Abeta.


Subject(s)
Amyloid beta-Peptides/pharmacology , Astrocytes/drug effects , Molecular Chaperones , Receptors, Lipoprotein/physiology , Animals , Apolipoproteins E/analysis , Cells, Cultured , Clusterin , Glycoproteins/analysis , Interleukin-1/genetics , Low Density Lipoprotein Receptor-Related Protein-1 , Rats , Rats, Sprague-Dawley , Receptors, Immunologic/physiology , Receptors, LDL/physiology
10.
J Biol Chem ; 274(42): 30001-7, 1999 Oct 15.
Article in English | MEDLINE | ID: mdl-10514484

ABSTRACT

Composition of central nervous system lipoproteins affects the metabolism of lipoprotein constituents within the brain. The epsilon4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer's disease via an unknown mechanism(s). As glia are the primary central nervous system cell type that synthesize apoE, we characterized lipoproteins secreted by astrocytes from wild type (WT), apoE (-/-), and apoE transgenic mice expressing human apoE3 or apoE4 in a mouse apoE (-/-) background. Nondenaturing size exclusion chromatography demonstrates that WT, apoE3, and apoE4 astrocytes secrete particles the size of plasma high density lipoprotein (HDL) composed of phospholipid, free cholesterol, and protein, primarily apoE and apoJ. However, the lipid:apoE ratio of particles containing human apoE is significantly lower than WT. ApoE localizes across HDL-like particle sizes. ApoJ localizes to the smallest HDL-like particles. ApoE (-/-) astrocytes secrete little phospholipid or free cholesterol despite comparable apoJ expression, suggesting that apoE is required for normal secretion of astrocyte lipoproteins. Further, particles were not detected in apoE (-/-) samples by electron microscopy. Nondenaturing immunoprecipitation experiments indicate that apoE and apoJ reside predominantly on distinct particles. These studies suggest that apoE expression influences the unique structure of astrocyte lipoproteins, a process further modified by apoE species.


Subject(s)
Apolipoproteins E/genetics , Astrocytes/metabolism , Lipoproteins/metabolism , Animals , Astrocytes/ultrastructure , Cells, Cultured , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Humans , Lipoproteins/isolation & purification , Lipoproteins/ultrastructure , Mice , Mice, Transgenic , Microscopy, Electron
11.
J Lipid Res ; 40(6): 1090-103, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10357841

ABSTRACT

Serum amyloid A (SAA) is an acute phase protein of unknown function that is involved in systemic amyloidosis and may also be involved in atherogenesis. The precise role of SAA in these processes has not been established. SAA circulates in plasma bound to high density lipoprotein-3 (HDL3). The pathway for the production of SAA-containing HDL is not known. To test whether apolipoprotein (apo)A-I-HDL is required in the production of SAA-HDL, we analyzed the lipopolysaccharide (LPS)-induced changes in apoA-I+/+ and apoA-I-/- mice. In apoA-I+/+ mice, after injection of LPS, remodeling of HDL occurred: total cholesterol increased and apoA-I decreased slightly and shifted to lighter density. Dense (density of HDL3) but large (size of HDL2 ) SAA-containing particles were formed. Upon fast phase liquid chromatography fractionation of plasma, >90% of SAA eluted with HDL that was enriched in cholesterol and phospholipid and shifted "leftward" to larger particles. Non-denaturing immunoprecipitation with anti-mouse apoA-I precipitated all of the apoA-I but not all of the SAA, confirming the presence of SAA-HDL devoid of apoA-I. In the apoA-I-/- mice, which normally have very low plasma lipid levels, LPS injection resulted in significantly increased total and HDL cholesterol. Greater than 90% of the SAA was lipid associated and was found on dense but large, spherical HDL particles essentially devoid of other apolipoproteins.We conclude that serum amyloid A (SAA) is able to sequester lipid, forming dense but large HDL particles with or without apoA-I or other apolipoproteins. The capacity to isolate lipoprotein particles containing SAA as the predominant or only apolipoprotein provides an important system to further explore the biological function of SAA.


Subject(s)
Acute-Phase Reaction , Apolipoprotein A-I/deficiency , Lipoproteins, HDL/blood , Serum Amyloid A Protein/analysis , Animals , Apolipoprotein A-I/genetics , Apolipoprotein A-I/metabolism , Cholesterol/blood , Chromatography, High Pressure Liquid , Immunosorbent Techniques , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Particle Size , Phospholipids/blood , Serum Amyloid A Protein/metabolism , Triglycerides/blood
12.
J Lipid Res ; 40(4): 753-63, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10191300

ABSTRACT

Apolipoprotein E plays a critical role in plasma lipoprotein clearance. Peptide models of a highly conserved, N-terminal domain of this protein have been shown to increase the binding of low density lipoprotein (LDL) to fibroblast cell surfaces independently of the low density lipoprotein receptor. Here we provide data to show that these peptides not only increase the binding of LDL, but also of high density lipoprotein, though not acetylated LDL. We also have data suggesting that this novel activity is mediated, at least in part, by a member of the scavenger receptor family, SR-AI. Furthermore, we show that this activity is also prominent in macrophages, a cell relevant to atherogenesis. In addition, this current paper provides evidence suggesting that this complex binding activity is initiated by a peptide-receptor interaction, and that our peptides are able to induce activity at physiologically relevant concentrations. This study provides evidence for a possible novel receptor interaction and further anti-atherogenic properties of apolipoprotein E and raises the possibility of a therapeutic potential of our peptide models.


Subject(s)
Apolipoproteins E/chemistry , Lipoproteins/metabolism , Membrane Proteins/analysis , Peptide Fragments/pharmacology , Receptors, Immunologic/metabolism , Receptors, Lipoprotein , Amino Acid Sequence , Animals , Blood , Cell Line , Chlorates/pharmacology , Egtazic Acid/pharmacology , Embryo, Mammalian , Fibroblasts , Heparin Lyase/pharmacology , Humans , Lipoproteins/blood , Lipoproteins, HDL/metabolism , Lipoproteins, LDL/metabolism , Macrophages/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mice , Models, Chemical , Molecular Sequence Data , Peptide Fragments/chemistry , Receptors, Scavenger , Scavenger Receptors, Class A , Scavenger Receptors, Class B
13.
J Lipid Res ; 39(7): 1372-81, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9684739

ABSTRACT

To examine the association of apolipoprotein (apo) E with nascent hepatic lipoproteins we have prepared stable transfectants of the rat hepatoma cell line McA RH7777 expressing the human apoE3 cDNA. When the nascent lipoproteins secreted from control cells were separated on fast protein liquid chromatography (FPLC) columns, rat apoE was detected in the very low density (VLDL) and high density lipoprotein (HDL) fractions, while rat apoA-I was found in the HDL and lipoprotein free fractions. Human apoE was also associated with the VLDL and HDL particles secreted from the transfected McA RH7777 cells. Expression of human apoE resulted in a significant decrease in the amount of rat apoA-I associated with the lipoprotein particles. Rat apoE was also displaced, but to a lesser extent. Infection of McA RH7777 cells at different multiplicities of infection with recombinant adenoviral vector containing the human apoE cDNA indicated that rat apoA-I was decreased in the HDL fractions at lower levels of expression of human apoE than was rat apoE. The HDL particles were further examined by immunoblotting of nondenaturing gradient gels and by non-denaturing immunoprecipitation. The results indicate that the high density lipoprotein (HDL) particles are heterogeneous in size and apolipoprotein composition with the majority of the rat and human apolipoproteins being located on different particles. These results suggest that the profile and concentration of HDL apolipoproteins produced in hepatocytes influences the assembly of the various subsets of secreted HDL.


Subject(s)
Apolipoproteins E/biosynthesis , Lipoproteins, HDL/biosynthesis , Lipoproteins, VLDL/biosynthesis , Animals , Apolipoprotein A-I/biosynthesis , Apolipoprotein A-I/isolation & purification , Apolipoproteins E/genetics , Apolipoproteins E/isolation & purification , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Humans , Lipoproteins, HDL/isolation & purification , Lipoproteins, VLDL/isolation & purification , Liver Neoplasms, Experimental , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Transfection , Tumor Cells, Cultured
14.
J Neurochem ; 70(5): 2070-81, 1998 May.
Article in English | MEDLINE | ID: mdl-9572293

ABSTRACT

Little is known about lipid transport and metabolism in the brain. As a further step toward understanding the origin and function of CNS lipoproteins, we have characterized by size and density fractionation lipoprotein particles from human CSF and primary cultures of rat astrocytes. The fractions were analyzed for esterified and free cholesterol, triglyceride, phospholipid, albumin, and apolipoproteins (apo) E, AI, AII, and J. As determined by lipid and apolipoprotein profiles, gel electrophoresis, and electron microscopy, nascent astrocyte particles contain little core lipid, are primarily discoidal in shape, and contain apoE and apoJ. In contrast, CSF lipoproteins are the size and density of plasma high-density lipoprotein, contain the core lipid, esterified cholesterol, and are spherical. CSF lipoproteins were heterogeneous in apolipoprotein content with apoE, the most abundant apolipoprotein, localized to the largest particles, apoAI and apoAII localized to progressively smaller particles, and apoJ distributed relatively evenly across particle size. There was substantial loss of protein from both CSF and astrocyte particles after density centrifugation compared with gel-filtration chromatography. The differences between lipoproteins secreted by astrocytes and present in CSF suggest that in addition to delivery of their constituents to cells, lipoprotein particles secreted within the brain by astrocytes may have the potential to participate in cholesterol clearance, developing a core of esterified cholesterol before reaching the CSF. Study of the functional properties of both astrocyte-secreted and CSF lipoproteins isolated by techniques that preserve native particle structure may also provide insight into the function of apoE in the pathophysiology of specific neurological diseases such as Alzheimer's disease.


Subject(s)
Astrocytes/metabolism , Lipoproteins/cerebrospinal fluid , Lipoproteins/metabolism , Animals , Animals, Newborn , Astrocytes/ultrastructure , Centrifugation, Density Gradient , Chromatography, Affinity , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Humans , Immunologic Techniques , Lipoproteins/ultrastructure , Microscopy, Electron , Rats , Rats, Sprague-Dawley
15.
J Neurosci ; 18(1): 195-204, 1998 Jan 01.
Article in English | MEDLINE | ID: mdl-9412500

ABSTRACT

Although the genetic link between the epsilon4 allele of apolipoprotein E (apoE) and Alzheimer's disease is well established, the isoform-specific activity of apoE underlying this correlation remains unclear. To determine whether apoE influences the neurotoxic actions of beta-amyloid (Abeta), we examined the effect of native preparations of apoE3 and E4 on Abeta-induced toxicity in primary cultures of rat hippocampal pyramidal neurons. The source of apoE was conditioned medium from HEK-293 cells stably transfected with human apoE3 or E4 cDNA. ApoE4 (10 microg/ml) alone was toxic to the cultures, whereas apoE3 had no effect. ApoE3 treatment prevented the toxicity induced by 10 microM Abeta(1-40) or Abeta(25-35). The apoE3 protective effect appears to be specific to Abeta-induced toxicity, because apoE3 did not protect against the cytotoxicity produced by NMDA or staurosporine, nor did apoE3 affect the increase in intracellular calcium induced by either NMDA or KCl. ApoE3 had no effect on the toxicity produced by Abeta in the presence of receptor-associated protein, an inhibitor of apoE receptors, particularly the LDL-receptor-related protein. Interaction with apoE receptors may not mediate the toxic actions of apoE4, because receptor-associated protein did not affect apoE4-induced neurotoxicity. Consistent with our previous biochemical experiments, analysis of the culture medium revealed that SDS-stable apoE3:Abeta complex is present in greater abundance than apoE4:Abeta complex. Thus, the protection from Abeta-induced neurotoxicity afforded by apoE3 treatment may result from clearance of the peptide by apoE3:Abeta complex formation and uptake by apoE receptors.


Subject(s)
Amyloid beta-Peptides/toxicity , Apolipoproteins E/pharmacology , Pyramidal Cells/cytology , Animals , Apolipoprotein E3 , Apolipoprotein E4 , Cell Death/drug effects , Cell Survival/drug effects , Cells, Cultured , Culture Media, Conditioned/pharmacology , Hippocampus/cytology , Humans , Kidney/cytology , Muscle Proteins/pharmacology , Neuroprotective Agents/pharmacology , Pyramidal Cells/chemistry , Pyramidal Cells/drug effects , Rats , Receptors, LDL/physiology
16.
J Neurosci Res ; 49(1): 9-18, 1997 Jul 01.
Article in English | MEDLINE | ID: mdl-9211985

ABSTRACT

In humans, apolipoprotein E (apoE) has three major isoforms, E2 (Cys112, Cys158), E3 (Cys112, Arg158), and E4 (Arg112, Arg158). While epsilon4 is a genetic risk factor for Alzheimer's disease (AD), epsilon2 may protect against late-onset AD. Using native preparations of apoE from conditioned tissue culture media or plasma lipoproteins, we have previously shown that when equivalent amounts of apoE3 or E4 were incubated with beta-amyloid (A beta), apoE3 formed 20 times as much SDS-stable complex with the peptide as apoE4. This preferential binding of A beta to apoE3 was abolished when apoE was purified by a process which includes delipidation and denaturation. Here we expand these observations to include A beta binding to lipoprotein-associated and purified apoE2. Lipoproteins isolated from the plasma of individuals homozygous for either epsilon2 or epsilon3 were incubated with A beta(1-40). SDS-stable complex formation was analyzed by a non-reducing gel shift assay, followed by immunoblotting with either A beta or apoE antibodies. ApoE2:A beta complex formation was comparable to apoE3:A beta in both native and purified preparations of apoE. In addition, lipoprotein-associated rat apoE (Arg112, Arg158), like human apoE4, did not form complex with A beta, while lipoprotein-associated rabbit apoE (Cys112, Arg158) did bind the peptide. These binding studies provide one possible explanation for protective effects of both apoE2 and E3 against the development of Alzheimer's disease.


Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Apolipoproteins E/metabolism , Peptide Fragments/metabolism , Alzheimer Disease/genetics , Animals , Apolipoprotein E2 , Apolipoprotein E3 , Apolipoproteins E/genetics , Apolipoproteins E/isolation & purification , Cell Line , Cysteine/chemistry , Disease Susceptibility , Genotype , Humans , Lipoproteins/blood , Lipoproteins/chemistry , Protein Binding , Rabbits , Rats , Species Specificity , Transfection
17.
J Biol Chem ; 271(11): 6062-70, 1996 Mar 15.
Article in English | MEDLINE | ID: mdl-8626391

ABSTRACT

Both apolipoprotein (apo) E and apoA-I are associated with lipoproteins, although with different particle classes. ApoE is associated with very low density lipoprotein (VLDL) and with the larger high density lipoprotein (HDL) subspecies, while apoA-I is found predominantly in association with most HDL subclasses. The genes encoding these proteins have a similar overall structure with the nucleotide sequences of the third and fourth exons coding for the mature protein. In an effort to understand the difference in lipoprotein association patterns of these two apoproteins, we have constructed and expressed chimeric apoproteins using cDNAs in which the third (n) and fourth (c) exons of human apoE and apoA-I are exchanged. McArdle rat hepatoma cells (McA-RH7777), which secrete VLDL- and HDL-like particles, were stably transfected with these cDNAs, and the cDNAs for human apoE and human apoA-I. Single spin NaBr gradient fractions of lipoprotein deficient serum-treated cell medium from transfected McA-RH7777 cells were analyzed. The distributions of transfected human apoE and apoA-I and endogenous rat apoE and apoA-I were compared with those of the chimeras. Among HDL subspecies, human apoE expressed by these cells is associated with particles of density 1.108 g/ml. Similarly, chimera apoA-InEc (exon 3 of apoA-I and exon 4 of apoE) is found in particles of density 1.111 g/ml. Human apoA-I, however, distributes in a broader range of particles with peak densities of 1.111 g/ml and 1.164 g/ml. The distribution of the complementary chimera, apoEnA-Ic, follows this same pattern, with peak particle densities of 1.098 and 1.137 g/ml. This is in contrast to the narrow distributions of endogenous rat apoE and apoA-I, which were found in particles of density 1.099 and 1.089 g/ml, respectively. When metabolically labeled medium was fractionated via gel filtration column chromatography, apoA-InEc was found to associate with the VLDL fractions; apoEnA-Ic was absent from these same fractions. These results suggest that the fourth exon largely determines the distinctive lipoprotein distribution patterns of these two human apoproteins and that the human apoA-I fourth exon sequence may account for the polydisperse HDL pattern as observed by others in transgenic mice expressing human apoA-I.


Subject(s)
Apolipoprotein A-I/metabolism , Apolipoproteins E/metabolism , Lipoproteins/metabolism , Amino Acid Sequence , Animals , Apolipoprotein A-I/genetics , Apolipoproteins E/genetics , Base Sequence , Cell Line , DNA, Complementary/genetics , Exons , Genetic Vectors , Humans , In Vitro Techniques , Lipoproteins, HDL/metabolism , Mice , Molecular Sequence Data , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Species Specificity , Transfection
18.
Metabolism ; 44(8): 1058-66, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7637648

ABSTRACT

These studies were conducted to determine relationships of plasma low-density lipoprotein (LDL) cholesterol concentrations and hepatic mRNA levels for apolipoprotein (apo) B, LDL receptor, and hepatic hydroxymethyl glutaryl coenzyme A (HMG CoA) synthase with plasma LDL apo B production and catabolic rates in baboons maintained on a low-cholesterol, low-fat chow diet and on a high-cholesterol, high-fat (HCHF) diet. Twelve baboons with LDL cholesterol levels ranging from low to high on the HCHF diet but with similar high-density lipoprotein (HDL) cholesterol levels were selected from a colony of selectively bred pedigreed baboons. LDL apo B turnover and hepatic mRNA concentrations for apo B, LDL receptor, and HMG CoA synthase were measured on a chow diet and again on a HCHF diet fed for 14 weeks. LDL apo B fractional catabolic rates decreased and production rates increased on the HCHF diet. Hepatic mRNA concentrations for apo B were not affected by the HCHF diet. Hepatic LDL receptor and HMG CoA synthase mRNA concentrations decreased on the HCHF diet as compared with the chow diet. LDL apo B fractional catabolic rate was negatively correlated with plasma cholesterol, LDL cholesterol, LDL apo B, and LDL apo B production and positively correlated with hepatic LDL receptor and HMG CoA synthase mRNA concentrations and with plasma LDL triglyceride to cholesterol ratio on the chow diet but not on the HCHF diet. LDL apo B production was positively correlated with plasma cholesterol, LDL cholesterol, and LDL apo B on the HCHF diet and negatively correlated with LDL triglyceride to cholesterol ratio on both chow and HCHF diets.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Apolipoproteins B/blood , Diet, Fat-Restricted/veterinary , Dietary Fats/administration & dosage , Lipoproteins, LDL/blood , Papio/metabolism , Animals , Apolipoproteins B/genetics , Apolipoproteins B/metabolism , Cholesterol, Dietary/administration & dosage , Cholesterol, LDL/blood , Female , Hydroxymethylglutaryl-CoA Synthase/analysis , Hydroxymethylglutaryl-CoA Synthase/genetics , Hydroxymethylglutaryl-CoA Synthase/physiology , Lipoproteins, LDL/metabolism , Liver/chemistry , Liver/enzymology , Male , RNA, Messenger/analysis , RNA, Messenger/genetics , Receptors, LDL/analysis , Receptors, LDL/genetics , Triglycerides/blood
19.
Biochim Biophys Acta ; 1262(2-3): 124-32, 1995 Jun 09.
Article in English | MEDLINE | ID: mdl-7599186

ABSTRACT

The mouse apolipoprotein (apo) E gene from strain C57BL/6 was isolated from a genomic DNA library and its complete nucleotide sequence, together with 1.3 kilobase of 5' flanking DNA and 300 base pairs of the 3' flanking DNA, was determined. Regulatory sequences in the proximal 5' flanking region of the gene were identified. Using a chloramphenicol acetyltransferase transient assay system, positive and negative cis-acting sequences were mapped within 380 base pairs of the 5' flanking region of the mouse apoE gene. Two nuclear protein binding sites were identified within this region by DNase I footprinting. We have characterized one of these regions, termed mouse apoE regulatory sequence (MARS-2), which spans nucleotides -151 to -133. Gel mobility shift assays using oligonucleotides of the MARS-2 sequence having specific deletions or substitutions as probes or competitors showed that the essential sequence of MARS-2 required for nuclear protein binding consists of 16 nucleotides encompassing -151 to -136. When nuclear extracts from different cells were examined, L cells and mouse liver nuclear protein contained the highest levels of binding protein for the MARS-2 probe. This protein, termed MARS-2 binding protein, was purified from mouse liver nuclear extracts to homogeneity using gel filtration and MARS-2 oligonucleotide-specific column chromatographic procedures. The Mr = 66,000 binding protein showed a gel mobility shift band that was identical to that of crude nuclear extracts.


Subject(s)
Apolipoproteins E/genetics , DNA-Binding Proteins/genetics , Animals , Base Sequence , DNA-Binding Proteins/isolation & purification , Deoxyribonuclease I , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Nuclear Proteins/isolation & purification , Promoter Regions, Genetic
20.
J Biol Chem ; 270(16): 9039-42, 1995 Apr 21.
Article in English | MEDLINE | ID: mdl-7721816

ABSTRACT

Apolipoprotein E (apoE), particularly the e4 allele, is genetically linked to the incidence of Alzheimer's disease. In vitro, apoE has been shown to bind beta-amyloid (A beta), an amyloidogenic peptide that aggregates to form the primary component of senile plaques. In previous work, we demonstrated that apoE3 from tissue culture medium binds to A beta with greater avidity than apoE4 (LaDu, M. J., Falduto, M. T., Manelli, A. M., Reardon, C. A., Getz, G. S., and Frail, D. E. (1994) J. Biol. Chem. 269, 23403-23406). This is in contrast to data using purified apoE isoforms as substrate for A beta (Strittmatter, W. J., Weisgraber, K. H., Huang, D. Y., Dong, L.-M., Salvesen, G. S., Pericak-Vance, M., Schmechel, D., Saunders, A. M., Goldgaber, D., and Roses, A. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8098-8102). Here we resolve this apparent discrepancy by demonstrating that the preferential binding of A beta to apoE3 is attenuated and even abolished with purification, a process that includes delipidation and denaturation. We compared the A beta binding capacity of unpurified apoE isoforms from both tissue culture medium and intact human very low density lipoproteins with that of apoE purified from these two sources. The interaction of human A beta-(1-40)-peptide and apoE was analyzed by nonreducing SDS-polyacrylamide gel electrophoresis followed by Western immunoblotting for either A beta or apoE immunoreactivity. While the level of the apoE3.A beta complex was approximately 20-fold greater compared with the apoE4.A beta complex in unpurified conditioned medium, apoE3 and apoE4 purified from this medium bound to A beta with comparable avidity. Moreover, using endogenous apoE on very low density lipoproteins from plasma of apoE3/3 and apoE4/4 homozygotes, apoE3 was again a better substrate for A beta than apoE4. However, apoE purified from these plasma lipoproteins exhibited little isoform specificity in binding to A beta. These results suggest that native preparations of apoE may be a more physiologically relevant substrate for A beta binding than purified apoE and further underscore the importance of subtle differences in apoE conformation to its biological activity.


Subject(s)
Amyloid beta-Peptides/metabolism , Apolipoproteins E/metabolism , Alzheimer Disease/etiology , Apolipoproteins E/isolation & purification , Culture Media , Humans , Lipoproteins, VLDL/metabolism , Protein Denaturation
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