ABSTRACT
Malnutrition is still considered endemic in many developing countries. Malnutrition-enteric infections may cause lasting deleterious effects on lipid metabolism, especially in children living in poor settings. The regional basic diet (RBD), produced to mimic the Brazilian northeastern dietary characteristics (rich in carbohydrate and low in protein) has been used in experimental malnutrition models, but few studies have explored the effect of chronic RBD on liver function, a central organ involved in cholesterol metabolism. This study aimed to investigate whether RBD leads to liver inflammatory changes and altered reverse cholesterol metabolism in C57BL6/J mice compared to the control group, receiving a standard chow diet. To evaluate liver inflammation, ionized calcium-binding adapter protein-1 (IBA-1) positive cell counting, interleukin (IL)-1ß immunohistochemistry, and tumor necrosis factor (TNF)-α and IL-10 transcription levels were analyzed. In addition, we assessed reverse cholesterol transport by measuring liver apolipoprotein (Apo)E, ApoA-I, and lecithin-cholesterol acyltransferase (LCAT) by RT-PCR. Furthermore, serum alanine aminotransferase (ALT) was measured to assess liver function. RBD markedly impaired body weight gain compared with the control group (P<0.05). Higher hepatic TNF-α (P<0.0001) and IL-10 (P=0.001) mRNA levels were found in RBD-challenged mice, although without detectable non-alcoholic fatty liver disease. Marked IBA-1 immunolabeling and increased number of positive-IBA-1 cells were found in the undernourished group. No statistical difference in serum ALT was found. There was also a significant increase in ApoA mRNA expression in the undernourished group, but not ApoE and LCAT, compared with the control. Altogether our findings suggested that chronic RBD-induced malnutrition leads to liver inflammation with increased ApoA-I activity.
Subject(s)
Apolipoprotein A-I/blood , Diet/adverse effects , Inflammation/metabolism , Malnutrition/metabolism , Animals , Apolipoprotein A-I/metabolism , Brazil , Chronic Disease , Humans , Inflammation/blood , Inflammation/pathology , Liver/metabolism , Male , Malnutrition/blood , Malnutrition/pathology , Mice , Mice, Inbred C57BLABSTRACT
Malnutrition is still considered endemic in many developing countries. Malnutrition-enteric infections may cause lasting deleterious effects on lipid metabolism, especially in children living in poor settings. The regional basic diet (RBD), produced to mimic the Brazilian northeastern dietary characteristics (rich in carbohydrate and low in protein) has been used in experimental malnutrition models, but few studies have explored the effect of chronic RBD on liver function, a central organ involved in cholesterol metabolism. This study aimed to investigate whether RBD leads to liver inflammatory changes and altered reverse cholesterol metabolism in C57BL6/J mice compared to the control group, receiving a standard chow diet. To evaluate liver inflammation, ionized calcium-binding adapter protein-1 (IBA-1) positive cell counting, interleukin (IL)-1β immunohistochemistry, and tumor necrosis factor (TNF)-α and IL-10 transcription levels were analyzed. In addition, we assessed reverse cholesterol transport by measuring liver apolipoprotein (Apo)E, ApoA-I, and lecithin-cholesterol acyltransferase (LCAT) by RT-PCR. Furthermore, serum alanine aminotransferase (ALT) was measured to assess liver function. RBD markedly impaired body weight gain compared with the control group (P<0.05). Higher hepatic TNF-α (P<0.0001) and IL-10 (P=0.001) mRNA levels were found in RBD-challenged mice, although without detectable non-alcoholic fatty liver disease. Marked IBA-1 immunolabeling and increased number of positive-IBA-1 cells were found in the undernourished group. No statistical difference in serum ALT was found. There was also a significant increase in ApoA mRNA expression in the undernourished group, but not ApoE and LCAT, compared with the control. Altogether our findings suggested that chronic RBD-induced malnutrition leads to liver inflammation with increased ApoA-I activity.
Subject(s)
Humans , Animals , Male , Rabbits , Rats , Apolipoprotein A-I/blood , Malnutrition/metabolism , Diet/adverse effects , Inflammation/metabolism , Brazil , Chronic Disease , Apolipoprotein A-I/metabolism , Malnutrition/pathology , Malnutrition/blood , Inflammation/pathology , Inflammation/blood , Liver/metabolism , Mice, Inbred C57BLABSTRACT
Stunning prior to slaughter is commonly used to render the animal insensible to pain. However, for certain markets, stunning is disallowed, unless the animal can fully recover if not slaughtered. There are very few available methods of inducing a fully recoverable stun. This paper describes the development of a microwave energy application system for stunning cattle. Cadaver heads were used to demonstrate that brain temperature could be raised to a point at which insensibility would be expected to occur (44°C), and to calculate the power and time combinations required to achieve this in a range of cattle weights. Surface heating was identified as a cause for potential concern, which was mitigated by the development of another type of microwave applicator. Although the applicator and process variables require validation in animal studies, this technology shows promise as a method of inducing a recoverable stun.
Subject(s)
Animal Welfare , Cattle , Microwaves , Pain/veterinary , Abattoirs , Animals , Cadaver , Hot Temperature , Pain/prevention & control , Unconsciousness/veterinaryABSTRACT
Intravenous (IV) iron is used to treat iron-deficiency anemia in patients with chronic kidney disease (CKD). Ferumoxytol is a novel iron formulation administered rapidly as two IV boluses of 510 mg each. In this placebo-controlled, double-blind, parallel-group study, 58 healthy volunteers received ferumoxytol in two 510 mg doses administered 24 h apart. Population pharmacokinetics (PK) analysis was conducted, and a two-compartment open model with zero-order input and Michaelis-Menten elimination was found to best describe the data. The population mean estimates for volume of distribution of the central compartment (V(1)), maximal elimination rate (V(max)), and ferumoxytol concentration at which rate of metabolism would be one-half of V(max) (K(m)) were 2.71 l, 14.3 mg/h, and 77.5 mg/l, respectively. When the effect of body weight on V(1) was added in the analysis, interindividual variability was found to be reduced. A noncompartmental analysis of two simulated 510-mg ferumoxytol doses was also performed to provide clinically interpretable data on half life and exposure. Ferumoxytol given as two consecutive 510-mg doses was well tolerated.
Subject(s)
Ferrosoferric Oxide/pharmacokinetics , Adolescent , Adult , Dose-Response Relationship, Drug , Female , Ferrosoferric Oxide/administration & dosage , Ferrosoferric Oxide/adverse effects , Half-Life , Humans , Indicators and Reagents , Magnetic Resonance Spectroscopy , Male , Middle Aged , Models, Statistical , Software , Young AdultABSTRACT
Several independent groups have reported targeted genomic editing in mammalian cells mediated by synthetic oligonucleotides. Nevertheless, the validity of data has been disputed because of experimental artefacts, inconsistent findings and low reproducibility. Here, we describe experiments designed to meet stringent criteria and completely eliminate artefactual results. In particular, by targeting cells expressing mutated enhanced green fluorescence protein (EGFP), which allow editing measurements at the protein level, and analyzing corrected clones by Southern blotting, we rigorously excluded spontaneous reversion, contamination artefacts, false-positives, or overestimation. Our findings provide unequivocal authentication that oligonucleotide-mediated gene editing is a real, not artefactual, phenomenon--a vital starting point from which to develop the technology into practical applications.
Subject(s)
Artifacts , Green Fluorescent Proteins/genetics , Oligonucleotides/genetics , Targeted Gene Repair/methods , Animals , Base Sequence/genetics , Blotting, Southern , CHO Cells , Clone Cells , Cricetinae , Cricetulus , Gene Targeting/methods , Oligonucleotides/pharmacology , Reproducibility of ResultsABSTRACT
Several procedures have been proposed to assess structural and functional characteristics of cryopreserved ram semen but none so far have yielded consistent relationships with in vivo fertility. The objectives of this study were to evaluate several sperm function tests as potential markers of in vivo ram fertility (determined by pregnancy rate in ewes) using frozen-thawed semen. In experiment 1, frozen-thawed straws (n=3 per ram) of semen from three high and three low fertility rams were assessed using fluorescent microscopy for (1) progressive motility, (2) viability and, (3) acrosomal status. In experiment 2, frozen-thawed straws (n=3 per ram) of semen from 18 rams of known fertility were analysed using either computer-assisted sperm analysis (CASA) for eight motion characteristics or flow cytometric staining for: (1) viability and acrosomal status, (2) plasma membrane status and capacitation-like changes, and (3) live cells following an osmotic resistance test (ORT). In experiment 3, platelet-activating factor (PAF) was isolated from straws (n=2 per ram) of semen using high-pressure liquid chromatography (HPLC) and quantified using HPLC-tandem mass spectrometry for 18 rams. In experiment 1, no association was found between motility, viability (% live) or acrosomal status (% damaged, % intact and % reacted) and in vivo fertility. In experiment 2, no correlation was found between motility (CASA), viability (% live), acrosomal status (% live, % live intact and % reacted), capacitation status (% capacitated, % non-capacitated), plasma membrane stability (% dead) and % live cells following ORT and ram in vivo fertility. In experiment 3, there was no relationship between PAF content in spermatozoa and ram fertility. In conclusion, we were unable to relate the in vivo fertility of rams with in vitro functional tests of their frozen-thawed semen and suggest that the fertility of a given semen sample cannot easily be quantified using available in vitro tests.
Subject(s)
Cervix Uteri , Fertility , Insemination, Artificial/veterinary , Semen Preservation/veterinary , Sheep/physiology , Spermatozoa/physiology , Animals , Cell Survival , Cryopreservation , Estrus Synchronization , Female , Hot Temperature , In Vitro Techniques , Insemination, Artificial/methods , Male , Platelet Activating Factor/analysis , Semen Preservation/methods , Sperm Capacitation , Sperm Motility , Spermatozoa/chemistryABSTRACT
Tigecycline, a novel glycylcycline, possesses broad-spectrum antimicrobial activity. A structural population pharmacokinetic model for tigecycline was developed based on data pooled from 5 phase I studies. Intravenous tigecycline was administered as single (12.5-300 mg) or multiple (25-100 mg) doses every 12 hours for up to 10 days. Three-compartment models with zero-order input and first-order elimination separately described the single- or multiple-dose full-profile data. Additional models were evaluated using a subset of the phase I data mimicking the phase II/III trial sparse-sampling scheme and dosage. A 2-compartment model best described the reduced phase I data following single or multiple doses and provided reliably accurate estimates of tigecycline AUC(0-12). This modeling supported phase II/III population pharmacokinetic model development to further determine individual patient tigecycline exposures for safety and efficacy analyses.
Subject(s)
Anti-Bacterial Agents/pharmacokinetics , Minocycline/analogs & derivatives , Adolescent , Adult , Aged , Anti-Bacterial Agents/blood , Area Under Curve , Clinical Trials, Phase I as Topic , Drug Administration Schedule , Female , Humans , Male , Middle Aged , Minocycline/blood , Minocycline/pharmacokinetics , Models, Biological , Multicenter Studies as Topic , TigecyclineABSTRACT
Tigecycline, a first-in-class expanded glycylcycline antimicrobial agent, has demonstrated efficacy in the treatment of complicated skin and skin structure infections (cSSSI) and complicated intra-abdominal (cIAI) infections. A population pharmacokinetic (PK) model for tigecycline was developed for patients with cSSSI or cIAI enrolled in two phase 2 clinical trials, and the influence of selected demographic factors and clinical laboratory measures was investigated. Tigecycline was administered as an intravenous loading dose followed by a 0.5- or 1-h infusion every 12 h for up to 14 days. Blood samples were collected the day before or the day of hospital discharge for the determination of serum tigecycline concentrations. Patient covariates were evaluated using stepwise forward (alpha = 0.05) and backward (alpha = 0.001) procedures. The predictive performance of the model was assessed separately using pooled data from either two phase 3 studies for patients with cSSSI or two phase 3 studies for patients with cIAI. A two-compartment model with zero-order input and first-order elimination adequately described the steady-state tigecycline concentration-time data. Tigecycline clearance was shown to increase with increasing weight, increasing creatinine clearance, and male gender (P < 0.001). The final model provided a relatively unbiased fit to each data set. Individual predicted values of the area under the concentration-time curve from 0 to 12 h (AUC(0-12)) were generally unbiased (median prediction error, -1.60% to -3.78%) and were similarly precise (median absolute prediction error, <4%) when compared across data sets. The population PK model provided the basis to obtain individual estimates of steady-state AUC(0-12) in later exposure-response analyses of tigecycline safety and efficacy in patients with cSSSI or cIAI.
Subject(s)
Abdomen , Anti-Bacterial Agents/pharmacokinetics , Bacterial Infections/metabolism , Minocycline/analogs & derivatives , Skin Diseases, Infectious/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Area Under Curve , Female , Humans , Male , Middle Aged , Minocycline/pharmacokinetics , Models, Statistical , Population , TigecyclineABSTRACT
Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 +/- 67.8 vs 271.7 +/- 20.1 micromol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10%) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.
Subject(s)
Erythrocytes/metabolism , Lipid Peroxidation , Liver Diseases, Parasitic/metabolism , Schistosomiasis mansoni/metabolism , Splenic Diseases/metabolism , Thiobarbituric Acid Reactive Substances/metabolism , Adolescent , Adult , Animals , Case-Control Studies , Child , Female , Follow-Up Studies , Humans , Liver Diseases, Parasitic/blood , Liver Diseases, Parasitic/parasitology , Male , Malondialdehyde/blood , Schistosoma mansoni , Schistosomiasis mansoni/complications , Schistosomiasis mansoni/surgery , Splenic Diseases/blood , Splenic Diseases/parasitology , Thiobarbituric Acid Reactive Substances/analysisABSTRACT
Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 æmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10 percent) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.
Subject(s)
Humans , Animals , Male , Female , Child , Adolescent , Adult , Erythrocytes , Lipid Peroxidation , Liver Diseases, Parasitic , Schistosoma mansoni , Schistosomiasis mansoni , Splenic Diseases , Thiobarbituric Acid Reactive Substances , Case-Control Studies , Follow-Up Studies , MalondialdehydeABSTRACT
Apolipoprotein E (apoE) is a multifunctional plasma glycoprotein involved in lipoprotein metabolism and a range of cell signalling phenomena. ApoE-deficient (apoE(-/-)) mice exhibit severe hypercholesterolaemia and are an excellent model of human atherosclerosis. ApoE somatic gene transfer and bone marrow transplantation in apoE(-/-) mice results in reversal of hypercholesterolaemia, inhibition of atherogenesis and regression of atherosclerotic plaque density. Replication defective adeno-associated virus vectors (rAAVs) are an attractive system currently in clinical trial for muscle-based heterologous gene therapy to express secreted recombinant plasma proteins. Here we have applied rAAV transduction of skeletal muscle to express wild-type (epsilon3) and a defective receptor-binding mutant (epsilon2) human apoE transgene in apoE(-/-) mice. In treated animals, apoE mRNA was present in transduced muscles and, although plasma levels of recombinant apoE fell below the detection levels of our ELISA (ie <10 ng/ml), circulating antibodies to human apoE and rAAV were induced. Up to 3 months after a single administration of rAAV/apoE3, a significant reduction in atherosclerotic plaque density in aortas of treated animals was observed (approximately 30%), indicating that low-level rAAV-mediated apoE3 expression from skeletal muscle can retard atherosclerotic progression in this well-defined genetic model.
Subject(s)
Apolipoproteins E/genetics , Arteriosclerosis/therapy , Dependovirus/genetics , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Animals , Antibodies/blood , Aorta/pathology , Apolipoproteins E/analysis , Apolipoproteins E/immunology , Arteriosclerosis/pathology , Blotting, Western/methods , Enzyme-Linked Immunosorbent Assay/methods , Humans , Mice , Mice, Knockout , Transduction, Genetic/methods , TransgenesABSTRACT
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 , TransfectionSubject(s)
Genetic Therapy , Alleles , Animals , Apolipoproteins/genetics , Arteriosclerosis/genetics , Arteriosclerosis/therapy , CHO Cells , Cricetinae , Humans , Lymphocytes/metabolism , Mice , Mice, Transgenic , Mutation , Polymorphism, Restriction Fragment Length , Polymorphism, Single NucleotideABSTRACT
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 , TransfectionSubject(s)
ATP-Binding Cassette Transporters/physiology , Up-Regulation , ATP Binding Cassette Transporter 1 , Cholesterol, HDL/blood , DNA-Binding Proteins , Humans , Liver X Receptors , Orphan Nuclear Receptors , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Retinoic Acid/physiology , Retinoid X Receptors , Transcription Factors/physiology , Transcription, Genetic , Up-Regulation/physiologyABSTRACT
High-density lipoproteins (HDL) have several antiatherogenic actions, including the ability to sequester cellular cholesterol, to protect low-density lipoproteins from oxidation and to inhibit platelet aggregation. An early event in atherogenesis is the adhesion and recruitment of blood monocytes, a process mediated by cell adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1) which is rapidly synthesized by endothelial cells in response to cytokines. It has been reported that HDL limits CAM expression in cultured human umbilical vein endothelial cells (HUVECs), implying that HDL also protects at an early stage in lesion development. Here, we have studied HDL suppression of CAM induction in human coronary artery endothelial cells (HCAECs), a model directly relevant to blood vessels susceptible to atherosclerosis. Arterial endothelial cells were preincubated with increasing amounts of total HDL, or different subfractions, and then activated with the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). Flow cytometric analysis failed to detect any downregulation of VCAM-1 or E-selectin expression by HDL in this model of vascular endothelium. Moreover, we were unable to confirm that HDL could suppress CAM induction in well-characterized, low-passage HUVECs, even though positive controls, 17beta-estradiol or a nitric oxide donor, did cause downregulation and factors such as variability in donors and HDL preparation, or culture conditions, were excluded. We tentatively conclude that, as isolated HDL did not downregulate CAM expression in cultured HCAECs or HUVECs, attenuation of CAM induction in arterial endothelium is unlikely to contribute to HDL antiatherogenic actions in vivo.
Subject(s)
Coronary Vessels/metabolism , Endothelium, Vascular/metabolism , Lipoproteins, HDL/physiology , Vascular Cell Adhesion Molecule-1/metabolism , Arteries/cytology , Arteries/metabolism , Cells, Cultured , Coronary Vessels/cytology , Down-Regulation , E-Selectin/metabolism , Endothelium, Vascular/cytology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Lipoproteins, HDL/bloodABSTRACT
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
Apolipoprotein A-I (apo A-I) and lecithin-cholesterol acyltransferase (LCAT) are constituents of circulating high-density lipoprotein (HDL) particles and play an important role in 'reverse cholesterol transport', the process by which cholesterol in peripheral tissues is transferred to the liver for excretion. Enhancing levels of apo A-I, as well as LCAT, in plasma may promote the removal of excess cholesterol from the arterial wall and thus reduce the formation of atherosclerotic lesions. Indeed, both apo A-I and LCAT genes have been identified as therapeutic targets to prevent or limit atherogenesis. Here, we have constructed two retroviral vectors, one containing LCAT cDNA and the neomycin phosphotransferase (NEO) gene (pLLEN), the other apo A-I cDNA, LCAT cDNA and the NEO gene (pLAPLEN) linked by internal ribosome entry sites (IRES). Both bi- and tricistronic retroviral vectors efficiently co-expressed their two or three genes when transfected into cultured mouse C2C12 muscle cells or human 293 cells. After 30 days, the retroviral vector sequences were retained by the host cells, whereas those of a conventional plasmid vector were lost. Moreover, transduced C2C12 mouse myoblasts maintained the ability for heterologous expression of human LCAT and apo A-I even after differentiation into myotubes. Stably-transduced clones of C2C12 cells were selected by neomycin (G418) resistance and continued to efficiently express human LCAT for 60 days. These findings indicate that the use of polycistronic retrovirus vectors to genetically modify myoblasts, which can be transplanted back into skeletal muscle, might be a safe and feasible strategy to express human apo A-I and LCAT and hence have therapeutic potential to regress atherosclerotic lesions.