ABSTRACT
Development of novel therapeutic agents aimed at treating neurodegenerative disorders such as Alzheimer's and Parkinson's diseases require chronic and preferentially oral dosing in appropriate preclinical rodent models. Since many of these disease models involve transgenic mice that are frequently aged and fragile, the commonly used oro-gastric gavage method of drug administration often confounds measured outcomes due to repeated stress and high attrition rates caused by esophageal complications. We employed a novel drug formulation in a peanut butter (PB) pellet readily consumed by mice and compared the stress response as measured by plasma corticosterone levels relative to oral administration via traditional gavage. Acute gavage produced significant elevations in plasma corticosterone comparable to those observed in mice subjected to stress-induced hyperthermia. In contrast, corticosterone levels following consumption of PB pellets were similar to levels in naive mice and significantly lower than in mice subjected to traditional gavage. Following sub-chronic administration, corticosterone levels remained significantly higher in mice subjected to gavage, relative to mice administered PB pellets or naive controls. Furthermore, chronic 30day dosing of a BACE inhibitor administered via PB pellets to PSAPP mice resulted in expected plasma drug exposure and Aß40 lowering consistent with drug treatment demonstrating target engagement. Taken together, this alternative method of oral administration by drug formulated in PB pellets results in the expected pharmacokinetics and pharmacodynamics with attenuated stress levels, and is devoid of the detrimental effects of repetitive oral gavage.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Arachis , Aspartic Acid Endopeptidases/antagonists & inhibitors , Chemistry, Pharmaceutical , Drug Delivery Systems/methods , Enzyme Inhibitors/pharmacology , Intubation, Gastrointestinal/adverse effects , Stress, Physiological/drug effects , Administration, Oral , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/blood , Animals , Aspartic Acid Endopeptidases/metabolism , Brain/metabolism , Corticosterone/blood , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacokinetics , Fever/blood , Male , Mice , Mice, Transgenic , Peptide Fragments/blood , Restraint, PhysicalABSTRACT
Sub-endothelial infiltration of monocytes occurs early in atherogenesis and is facilitated by cell adhesion molecules that are up-regulated on activated endothelium. Apolipoprotein E (apoE) helps protect against atherosclerosis, in part, because apoE particles secreted by macrophages have local beneficial effects at lesion sites. Here, we hypothesize that such protection includes anti-inflammatory actions and investigate whether cell-derived apoE can inhibit tumor necrosis factor-alpha-mediated up-regulation of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Two models were used to mimic endothelial exposure to macrophage-derived apoE. In the first, HUVECs were transiently transfected to secrete apoE; VCAM-1 induction inversely correlated with secretion of apoE into the media (r = -0.76, p < 0.001). In the second, incubation of HUVECs with media from recombinant Chinese hamster ovary (CHO) cells expressing apoE (CHO(apoE)) also reduced VCAM-1 in a dose-dependent manner (r = -0.70, p < 0.001). Characterization of CHO(apoE) cell-derived apoE revealed several similarities to apoE particles secreted by human blood monocyte-derived macrophages. The suppression of endothelial activation by apoE most likely occurs via stimulation of endothelial nitric oxide synthase; apoE increased levels of intracellular nitric oxide and its surrogate marker, cyclic guanosine monophosphate, while the nitric oxide synthase inhibitor, ethyl-isothiourea, blocked its effect. We propose that apoE secreted locally at lesion sites by macrophages may be anti-inflammatory by stimulating endothelium to release NO and suppress VCAM-1 expression.
Subject(s)
Apolipoproteins E/physiology , Down-Regulation/physiology , Endothelium, Vascular/metabolism , Vascular Cell Adhesion Molecule-1/genetics , Animals , CHO Cells , Cricetinae , Endothelium, Vascular/cytology , Humans , Nitric Oxide/metabolism , TransfectionABSTRACT
Recent epidemiological studies show a reduced prevalence of Alzheimer's disease (AD) in patients treated with inhibitors of cholesterol biosynthesis. Moreover, the cholesterol-transport protein, apolipoprotein E4, and elevated cholesterol are important risk factors for AD. Additionally, in vitro and in vivo studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein (APP) to beta-amyloid, the major constituent of senile plaques. Cholesterol plays a crucial role in maintaining lipid rafts in a functional state. Lipid rafts are cholesterol-enriched membrane microdomains implicated in signal transduction, protein trafficking, and proteolytic processing. Since APP, beta-amyloid, and the putative gamma-secretase, presenilin-1 (PS-1), have all been found in lipid rafts, we hypothesized that the recently identified beta-secretase, Asp2 (BACE1), might also be present in rafts. Here, we report that recombinant Asp2 expressed in three distinct cell lines is raft associated. Using both detergent and nondetergent methods, Asp2 protein and activity were found in a light membrane raft fraction that also contained other components of the amyloidogenic pathway. Immunoisolation of caveolin-containing vesicles indicated that Asp2 was present in a unique raft population distinct from caveolae. Finally, depletion of raft cholesterol abrogated association of Asp2 with the light membrane fraction. These observations are consistent with the raft localization of APP processing and suggest that the partitioning of Asp2 into lipid rafts may underlie the cholesterol sensitivity of beta-amyloid production.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Caveolins/metabolism , Membrane Microdomains/enzymology , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases , Amyloid beta-Protein Precursor/metabolism , Animals , Carbonates/chemistry , Caveolae/chemistry , Caveolae/metabolism , Cell Fractionation , Cell Line , Cholesterol/metabolism , Detergents , Endopeptidases , Humans , Membrane Microdomains/chemistry , Membrane Proteins/metabolism , Microscopy, Fluorescence , Peptide Fragments/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
The LDL receptor (LDL-R) promotes the specific endocytosis and lysosomal delivery of extracellular lipoprotein ligands via clathrin-coated pits. It was widely assumed that other closely related members of the LDL-R gene family would have similar functions, but recent experimental evidence has revealed that one such protein, apolipoprotein E receptor 2 (apoER2), has a critical role as an "outside-in" signal transducer in the brain. ApoER2 signaling appears to require interaction between its cytoplasmic domain and adapter molecules such as Dab1, JIP 1 and JIP 2, and PSD-95. Many of the receptors for other signaling pathways affected by such adapter molecules are compartmentalized into specialized microdomains within the plasma membrane termed caveolae. Here, we show that apoER2, but not LDL-R, is localized to caveolae, supporting the concept that its physiological role is in cell signaling, rather than in endocytosing ligands.
Subject(s)
Caveolae/metabolism , Cell Membrane/metabolism , Receptors, Lipoprotein/biosynthesis , Alternative Splicing , Animals , CHO Cells , Caveolin 1 , Caveolins/biosynthesis , Cricetinae , Detergents/pharmacology , Endocytosis , Immunoblotting , LDL-Receptor Related Proteins , Ligands , Models, Genetic , Precipitin Tests , Protein Structure, Tertiary , Receptors, LDL/biosynthesis , Receptors, Lipoprotein/chemistry , Recombinant Proteins/metabolism , Signal Transduction , Subcellular FractionsABSTRACT
Apolipoprotein (apo) E is a polymorphic plasma protein, synthesized mainly by liver. Here, we evaluate whether synthetic DNA-RNA oligonucleotides (chimeraplasts) can convert a dysfunctional isoform, apoE2 (C --> T, R158C), which causes Type III hyperlipidemia and premature atherosclerosis, into apoE3. First, we treated recombinant Chinese hamster ovary cells stably secreting apoE2 with a 68-mer apoE2 to apoE3 chimeraplast. About one-third of apoE2 was converted to apoE3, and the repair was stable through 12 passages. Subcloning treated cells produced both apoE2 and apoE3 clones. Direct sequencing and reverse transcription polymerase chain reaction confirmed the genotype, whereas phenotypic change was verified by isoelectric focusing and immunoblotting of secreted proteins. Second, we established that the APOE2 gene can be targeted both in vivo, using transgenic mice overexpressing human apoE2, and in chromosomal context, using cultured lymphocytes from a patient homozygous for the epsilon2 allele. We conclude that chimeraplasty has the potential to convert the apoE2 mutation in patients with Type III hyperlipidemia to apoE3.
Subject(s)
Apolipoproteins E/genetics , Lymphocytes/physiology , Amino Acid Substitution , Animals , Apolipoprotein E2 , Apolipoprotein E3 , Apolipoproteins E/chemistry , Base Sequence , CHO Cells , Cricetinae , Crosses, Genetic , Female , Genetic Therapy , Genomic Library , Genotype , Humans , Lymphocytes/cytology , Male , Mice , Mice, Transgenic , Oligodeoxyribonucleotides , Oligoribonucleotides , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , RNA, Messenger/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
Apolipoprotein-E (apoE) protects against coronary artery disease via hepatic removal of atherogenic remnant lipoproteins, sequestration of cholesterol from vessel walls and local anti-oxidant, anti-platelet and anti-inflammatory actions. ApoE gene transfer may thus ameliorate a hyperlipidaemic profile and have beneficial effects at lesion sites to prevent or regress atherosclerosis, a concept endorsed by adenoviral-mediated hepatic expression studies. Here, using plasmid vectors expressing allelic human apoE2 or apoE3 isoforms, skeletal muscle was evaluated as an effective secretory platform for apoE gene augmentation. Transfected myoblasts and myotubes were found to efficiently secrete recombinant apoE in vitro as spherical 10-16 nm lipoprotein particles with pre-beta mobility. Intramuscular plasmid injection in apoE(-/-) mice, which develop spontaneous atherosclerotic plaque and xanthoma resulted in expression and secretion of apoE. Human apoE mRNA was detected by RT-PCR in injected muscles and, although concentrations of apoE3, which is rapidly cleared from plasma, were near ELISA detection limits, levels of plasma apoE2 were measurable (17.5 +/- 4.3 ng/ml). To assess whether muscle-based expression of apoE2 could inhibit atherogenesis, long-term follow-up studies were conducted. Although hyperlipidaemia was not reduced in treated animals, end-point pathology showed clear retardation of atherosclerotic and xanthomatous lesions. Up to 9 months following a single apoE2 plasmid administration, atherosclerotic lesion coverage in proximal aorta was significantly reduced by 20-30% (P < 0.01), whereas development of gross dorsal xanthoma (>5 mm diameter) was effectively reduced to zero. We conclude that expression of apoE from ectopic muscle sites has therapeutic potential to limit progression of atherosclerosis.
Subject(s)
Apolipoproteins E/genetics , Arteriosclerosis/therapy , Genetic Therapy , Muscle, Skeletal/metabolism , Plasmids , Xanthomatosis/therapy , Animals , Apolipoprotein E2 , Apolipoprotein E3 , Apolipoproteins E/blood , Apolipoproteins E/deficiency , Apolipoproteins E/metabolism , Cell Line , Disease Progression , Gene Expression , Gene Transfer Techniques , Genetic Vectors , Humans , Hyperlipidemias/metabolism , Hyperlipidemias/therapy , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Recently, we reported that apoE inhibits platelet reactivity by stimulating NO release and postulated apoE-receptor activation of intracellular NO synthase (eNOS). Here, we implicate a low density lipoprotein receptor (LDL-R) family member by studying ligand requirements using purified apoE isoforms, synthetic peptides, and the receptor antagonist, receptor-associated protein (RAP). Then, using a homology cloning approach and degenerate PCR primers to amplify the conserved Cys-rich binding domain of the LDL-R family, this receptor was identified as LRP8 (formerly termed, apoER2), a newly described brain protein with several splice variants. Immunoprecipitation of platelet membranes with anti-peptide antisera confirmed protein expression, while analysis of RNA from platelets and two megakaryocytic cell lines (Meg-01 and HEL) disclosed that the major LRP8 transcript lacked binding repeats 4-6 (LRP8delta4-6) but contained the full-length cytoplasmic tail. Sequence analysis of cytoplasmic LRP8 revealed several peptide motifs with potential for cellular signaling and we propose this as a rational mechanism through which apoE inhibits platelet aggregation.
Subject(s)
Blood Platelets/metabolism , Receptors, Lipoprotein/blood , Receptors, Lipoprotein/genetics , Alternative Splicing , Amino Acid Sequence , Apolipoproteins E/chemistry , Apolipoproteins E/pharmacology , Binding Sites , Cell Line , Cell Membrane/metabolism , Conserved Sequence , Cysteine , Humans , Hyperlipidemias/blood , Hyperlipidemias/genetics , Kinetics , LDL-Receptor Related Proteins , Lipoproteins, VLDL/blood , Low Density Lipoprotein Receptor-Related Protein-1 , Megakaryocytes/metabolism , Peptide Fragments/chemistry , Platelet Aggregation , RNA, Messenger/genetics , Receptors, Immunologic/metabolism , Receptors, Lipoprotein/chemistry , Transcription, GeneticABSTRACT
Platelets are small cells, 1/14th the volume of erythrocytes, and about 1000 billion circulate in human blood as smooth anucleate disks. Their job is to survey the lining of our blood vessels, the endothelium. In acute damage and extravasation, platelets are activated by contact with exposed collagen and aggregate together at the wound sites to initiate clotting and stop bleeding. Forming a physical plug to seal a hemorrhaging vessel is the key role of blood platelets. However, milder injury to the endothelium, perhaps a result of high blood pressure, raised plasma cholesterol, or smoking, also causes platelets to adhere to the internal walls of arteries. Such precipitate adhesion and activation of platelets initiates an inflammatory response of the vessel wall and predisposes to vascular complications, including thrombosis, premature heart disease, myocardial infarcts or strokes, and diabetes. It is essential, therefore, that during normal vascular hemostasis platelet activation is tightly controlled. Indeed, both platelets and endothelial cells produce and secrete chemicals that directly inhibit platelet aggregation. A key agent is the free radical gas nitric oxide (NO). Here, we review how this 30-Da molecular messenger is synthesized by a catalytic cassette 10,000 times larger and how it functions to suppress platelet "stickiness." We also present new evidence that directly links plasma lipoproteins with platelet activation: we describe at the molecular level how apoE, a protein with a prominent role in cholesterol transport, interacts with the platelet surface to stimulate NO production and hence attenuate platelet activation.
Subject(s)
Nitric Oxide , Platelet Aggregation , Apolipoproteins E/pharmacology , Blood Platelets/physiology , Cyclic GMP/biosynthesis , Humans , Lipoproteins, HDL/pharmacology , Nitric Oxide/physiology , Nitric Oxide Synthase/blood , Platelet Aggregation Inhibitors/pharmacologySubject(s)
Blood Platelets/chemistry , Blood Platelets/metabolism , Receptors, Immunologic/blood , Cell Membrane/chemistry , Electrophoresis, Polyacrylamide Gel , Humans , Low Density Lipoprotein Receptor-Related Protein-1 , Receptors, LDL/blood , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
Atherosclerotic plaques develop in the arterial wall from complex multicellular processes following the early recruitment of circulating monocytes. Infiltration of monocytes is mediated by cell adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1) which is rapidly induced in endothelial cells in response to cytokines. Apolipoprotein E (apo E), a 34-kDa polypeptide, helps protect against atherosclerosis, in part, because apo E phospholipid particles secreted by macrophages may have local protective effects within lesions. Here we have investigated whether purified plasma apo E, complexed with dimyristoyl phosphatidylcholine (DMPC) vesicles, can inhibit cytokine-induced vascular cell adhesion molecule-1 (VCAM-1) expression in human umbilical vein endothelial cells (HUVECs). Expression of VCAM-1 in endothelial cells after exposure to tumour necrosis factor-alpha (TNF-alpha) or interleukin 1beta (IL-1beta) was quantified by ELISA and shown to be partially inhibited by 17beta-estradiol (40-60% inhibition) or by S-nitroso-L-glutathione, a nitric oxide donor (20-25%). However, preincubations with physiological concentrations (10-100 microg protein/ml) of apo E DMPC did not downregulate VCAM-1 expression, even with extended preincubation times. These findings were confirmed using a fluorescence-activated cell sorter (FACS) for analysis which indicated additionally that apo E-DMPC had no effect on sub-populations within the HUVEC cultures. Finally, apo E-DMPC vesicles were also unable to suppress TNF-alpha-induced upregulation of E-selectin or intercellular adhesion molecule-1 (ICAM-1). We conclude that plasma apo E is unlikely to be important in limiting endothelial activation.
Subject(s)
Apolipoproteins E/physiology , Cell Adhesion Molecules/biosynthesis , Cytokines/pharmacology , Endothelium, Vascular/metabolism , Apolipoproteins E/administration & dosage , Apolipoproteins E/pharmacology , Cell Separation , Cells, Cultured , Dimyristoylphosphatidylcholine , E-Selectin/biosynthesis , Endothelium, Vascular/drug effects , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Intercellular Adhesion Molecule-1/biosynthesis , Interleukin-1/pharmacology , Liposomes , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation , Vascular Cell Adhesion Molecule-1/biosynthesisABSTRACT
We have previously reported that plasma apolipoprotein (apo) E-containing high density lipoprotein particles have a potent anti-platelet action, apparently by occupying saturable binding sites in the cell surface. Here we show that purified apoE (10-50 microg/ml), complexed with phospholipid vesicles (dimyristoylphosphatidylcholine, DMPC), suppresses platelet aggregation induced by ADP, epinephrine, or collagen. This effect was not due to sequestration of cholesterol from platelet membranes; apoE x DMPC chemically modified with cyclohexanedione (cyclohexanedione-apoE x DMPC) did not inhibit aggregation but nevertheless removed similar amounts of cholesterol as untreated complexes, about 2% during the aggregation period. Rather we found that apoE influenced intracellular platelet signaling. Thus, apoE x DMPC markedly increased cGMP in ADP-stimulated platelets which correlated with the resulting inhibition of aggregation (r = 0.85; p < 0.01, n = 10), whereas cyclohexanedione-apoE x DMPC vesicles had no effect. One important cellular mechanism for up-regulation of cGMP is through stimulation of nitric oxide (NO) synthase, the NO generated by conversion of L-arginine to L-citrulline, binds to and activates guanylate cyclase. This signal transduction pathway was implicated by the finding that NO synthase inhibitors of distinct structural and functional types all reversed the anti-platelet action of apoE, whereas a selective inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (100 nM), had a similar reversing action. Direct confirmation that apoE stimulates NO synthase was obtained by use of L-[3H]arginine; platelets pretreated with apoE x DMPC produced markedly more L-[3H]citrulline (0.71 +/- 0.1 pmol/h/10(9) platelets) than controls (0.18 +/- 0.03; p < 0.05). In addition, hemoglobin which avidly binds NO also suppressed the anti-aggregatory effect, indicating that apoE stimulated sufficient production of NO by platelets for extracellular release to occur. We conclude that apoE inhibits platelet aggregation through the L-arginine:NO signal transduction pathway.
Subject(s)
Apolipoproteins E/pharmacology , Arginine/physiology , Platelet Aggregation Inhibitors , Platelet Aggregation/drug effects , 1-Methyl-3-isobutylxanthine/pharmacology , Blood Platelets/enzymology , Cholesterol/metabolism , Cyclic AMP/blood , Cyclic GMP/blood , Dimyristoylphosphatidylcholine , Enzyme Inhibitors/pharmacology , Guanylate Cyclase/antagonists & inhibitors , Humans , Lipoproteins, HDL/pharmacology , Membrane Lipids/metabolism , Nitric Oxide/physiology , Nitric Oxide Synthase/blood , Oxadiazoles/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Quinoxalines/pharmacologySubject(s)
Apolipoproteins E/pharmacology , Lipoproteins, HDL/pharmacology , Platelet Aggregation/drug effects , Apolipoproteins E/blood , Apolipoproteins E/isolation & purification , Chromatography, Affinity , Epinephrine/pharmacology , Humans , In Vitro Techniques , Lipoproteins, HDL/blood , Lipoproteins, HDL/isolation & purification , Liver Cirrhosis/bloodABSTRACT
We have previously shown that plasma HDL-E, a minor subclass of high-density lipoproteins (HDL) containing apolipoprotein (apo) E, has a potent anti-platelet effect and implicated apoE as the active constituent. Recently, apoE complexes with phospholipids (DMPC) were reported to inhibit thrombin-induced aggregation by sequestering platelet membrane cholesterol. Here we demonstrate that platelet cholesterol depletion is an improbable explanation for the suppressive effect of apoE:DMPC on ADP-mediated platelet aggregation; only 0.5% of cholesterol was released prior to addition of ADP to initiate aggregation while lactoferrin, which does not accept cellular cholesterol, was also inhibitory. Previous studies have shown that apoE and lactoferrin are both bound by platelets but whether this provides the initial stimulus for suppression of aggregation remains to be established.
Subject(s)
Adenosine Diphosphate/pharmacology , Apolipoproteins E/pharmacology , Blood Platelets/metabolism , Cell Membrane/metabolism , Cholesterol/metabolism , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Animals , Apolipoprotein A-I/pharmacology , Blood Platelets/drug effects , Cell Membrane/drug effects , Dimyristoylphosphatidylcholine , Humans , Liposomes , RabbitsABSTRACT
We have previously shown that plasma HDL-E, a minor subclass of high-density lipoproteins (HDL) containing apolipoprotein (apo) E, has a potent anti-platelet effect and implicated apoE as the active constituent. Recently, apoE complexes with phospholipids (DMPC) were reported to inhibit thrombin-induced aggregation by sequestering platelet membrane cholesterol. Here we demonstrate that platelet cholesterol depletion is an improbable explanation for the suppressive effect of apoE:DMPC on ADP-mediated platelet aggregation; only 0.5% of cholesterol was released prior to addition of ADP to initiate aggregation while lactoferrin, which does not accept cellular cholesterol, was also inhibitory. Previous studies have shown that apoE and lactoferrin are both bound by platelets but whether this provides the initial stimulus for suppression of aggregation remains to be established.