The combined effect of pravastatin and aspirin on clinical cardiovascular events

The 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, can achieve significant reductions in plasma low-density lipoprotein (LDL)-cholesterol levels. Experimental and clinical evidence now shows that some statins interfere with formation of atherosclerotic lesions independent of their hypolipidemic properties. Vulnerable plaque rupture can result in thrombus formation and artery occlusion; this plaque deterioration is responsible for most acute coronary syndromes, including myocardial infarction (MI), unstable angina, and coronary death, as well as coronary heart diseaseequivalent non-hemorrhagic stroke. Inhibition of HMG-CoA reductase has potential pleiotropic effects other than lipid-lowering, as statins block mevalonic acid production, a precursor to cholesterol and numerous other metabolites. Statins' beneficial effects on clinical events may also thus involve nonlipid-related mechanisms that modify endothelial function, inflammatory responses, plaque stability, and thrombus formation. Aspirin, routinely prescribed to post-MI patients as adjunct therapy, may potentiate statins beneficial effects, as aspirin does not compete metabolically with statins but acts similarly on atherosclerotic lesions. Common functions of both medications include inhibition of platelet activity and aggregation, reduction in atherosclerotic plaque macrophage cell count, and prevention of atherosclerotic vessel endothelial dysfunction. The Cholesterol and Recurrent Events (CARE) trial provides an ideal population in which to examine the combined effects of pravastatin and aspirin. Lipid levels, intermediate outcomes, are examined by pravastatin and aspirin status, and differences between the two pravastatin groups are found. A modified Cox proportional-hazards model with aspirin as a time-dependent covariate was used to determine the effect of aspirin and pravastatin on the clinical cardiovascular composite endpoint of coronary heart disease death, recurrent MI or stroke. Among those assigned to pravastatin, use of aspirin reduced the composite primary endpoint by 35%; this result was similar by gender, race, and diabetic status. Older patients demonstrated a nonsignificant 21% reduction in the primary outcome, whereas the younger had a significant reduction of 43% in the composite primary outcome. Secondary outcomes examined include coronary artery bypass graft (38% reduction), nonsurgical bypass, peripheral vascular disease, and unstable angina. Pravastatin and aspirin in a post-MI population was found to be a beneficial combination that seems to work through lipid and nonlipid, anti-inflammatory mechanisms. ^

Genome-wide association study of LDL-cholesterol over ApoB ratio

Genome-Wide Association Study analytical (GWAS) methods were applied in a large biracial sample of individuals to investigate variation across the genome for its association with a surrogate low-density lipoprotein (LDL) particle size phenotype, the ratio of LDL-cholesterol level over ApoB level. Genotyping was performed on the Affymetrix 6.0 GeneChip with approximately one million single nucleotide polymorphisms (SNPs). The ratio of LDL cholesterol to ApoB was calculated, and association tests used multivariable linear regression analysis with an additive genetic model after adjustment for the covariates sex, age and BMI. Association tests were performed separately in African Americans and Caucasians. There were 9,562 qualified individuals in the Caucasian group and 3,015 qualified individuals in the African American group. Overall, in Caucasians two statistically significant loci were identified as being associated with the ratio of LDL-cholesterol over ApoB: rs10488699 (p<5 x10-8, 11q23.3 near BUD13) and the SNP rs964184 (p<5 x10-8 11q23.3 near ZNF259). We also found rs12286037 ((p<4x10-7) (11q23.3) near APOA5/A4/C3/A1 with suggestive associate in the Caucasian sample. In exploratory analyses, a difference in the pattern of association between individuals taking and not taking LDL-cholesterol lowering medications was observed. Individuals who were not taking medications had smaller p-value than those taking medication. In the African-American group, there were no significant (p<5x10-8) or suggestive associations (p<4x10-7) with the ratio of LDL-cholesterol over ApoB after adjusting for age, BMI, and sex and comparing individuals with and without LDL-cholesterol lowering medication. Conclusions: There were significant and suggestive associations between SNP genotype and the ratio of LDL-cholesterol to ApoB in Caucasians, but these associations may be modified by medication treatment.^

THE NEW ROLE OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9: A CONNECTION OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9, APOLIPOPROTEIN B, and AUTOPHAGY

Plasma low-density lipoprotein (LDL) levels are positively correlated with the incidence of coronary artery disease. In the circulation, the plasma LDL clearance is mainly achieved by the uptake via LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a newly discovered gene, playing an important role in LDL metabolism. Gain-of-function mutations of PCSK9 lead to hypercholesterolemia and loss-of-function mutations of PCSK9 are associated with decrease of LDL cholesterol. The effects of PCSK9 on cholesterol levels are the consequence of a strong interaction between the catalytic domain of PCSK9 and epidermal growth factor-like repeat A (EGF-A) domain of LDLR on the cell surface of hepatocytes. This PCSK9/LDLR complex enters the cell via endocytosis, where both PCSK9 and LDLR are removed via the lysosome pathway, resulting in decreased levels of LDLR and accumulation of LDL in the plasma. However, whether this is the exclusive function of PCSK9 on LDL metabolism was challenged by us; we observed PCSK9 interacted with apolipoprotein B (apoB) and increased apoB production, irrespective of the LDLR. ApoB is the primary structure protein of LDL particle and it also serves as the ligand for the LDL receptor. There is ample evidence showing that the levels of apoB are a better indicator for heart disease than either total cholesterol or LDL cholesterol levels. We used a second-generation adenoviral vector to overexpress PCSK9 (Ad-PCSK9) in wild-type C57BL/6 and LDLR deficient mice (Ldlr-/- and Ldlr-/-Apobec1-/-). Our study revealed that overexpression of PCSK9 promoted the production and secretion of apoB in the form of very-low density lipoprotein (VLDL), which is the precursor of LDL, in the 3 mouse models studied (C57BL/6J, Ldlr-/-, and Ldlr-/-Apobec1-/-). The increased apoB production in mice was regulated at post-transcriptional levels, since there was no difference in apoB mRNA levels between mice treated with Ad-PCSK9 and control vector Ad-Null. By using pulse-chase experiment on primary hepatocytes, we showed that overexpression of PCSK9 increased the secretion of apoB, independent of LDLR. In the circulation, we showed that PCSK9 was associated with LDL particles. By using 3 different protein–protein interaction assays of co-immunoprecipitation, mammalian two-hybrid system, and in situ proximity ligation assay, we demonstrated a direct protein–protein interaction between PCSK9 and apoB. The impact of this interaction inhibited the physiological removal process of apoB via autophagosome/lysosome pathway in an LDLR-independent fashion, resulting in increased production and secretion of apoB-containing lipoproteins. The significance of this process was shown in the Pcsk9 knockout mice in the background of Ldlr-/-Apobec1-/- mice (triple knockout mice); in the absence of Pcsk9 (triple knockout mice) the levels of cholesterol, triacylglycerol, and apoB decreased significantly in comparison to that of Ldlr-/-Apobec1-/- mice. Taken together, our study demonstrated a direct intracellular interaction of PCSK9 with apoB, resulting in the inhibition of apoB degradation via the autophagosome/lysosome pathway independent of LDLR. This discovery provides a new concept of the importance of PCSK9 and suggests new approaches for the therapeutic intervention of hyperlipidemia.

NOVEL THERAPEUTIC TARGETS IDENTIFIED BY HIGH-THROUGHPUT TECHNOLOGIES FOR TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancers (TNBC) are characterized by the lack of or reduced expression of the estrogen and progesterone receptors, and normal expression of the human epidermal growth factor receptor 2. The lack of a well-characterized target for treatment leaves only systemic chemotherapy as the mainstay of treatment. Approximately 60-70% of patients are chemosensitive, while the remaining majority does not respond. Targeted therapies that take advantage of the unique molecular perturbations found in triple-negative breast cancer are needed. The genes that are frequently amplified or overexpressed represent potential therapeutic targets for triple-negative breast cancer. The purpose of this study was to identify and validate novel therapeutic targets for triple-negative breast cancers. 681 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in 2 data sets. For two genes, 3 of the 4 siRNAs showed preferential growth inhibition in TNBC cells. These two genes were the low density lipoprotein receptor-related protein 8 (LRP8) and very low-density lipoprotein receptor (VLDLR). Exposure to their cognate ligands, reelin and apolipoprotein E isoform 4 (ApoE4), stimulated the growth of TNBC cells in vitro. Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. High-throughput protein and metabolic arrays revealed that ApoE4 stimulation rescued TNBC cells from serum-starvation induced up-regulation of genes involved in lipid biosynthesis, increased protein expression of oncogenes involved in the MAPK/ERK and DNA repair pathways, and reduced the serum-starvation induction of biochemicals involved in oxidative stress response and glycolytic metabolism. shLRP8 MDA-MB-231 xenografts had reduced tumor volume, in comparison to parental and shCON xenografts. These results indicate that LRP8-APOE signaling confers survival advantages to TNBC tumors under reduced nutrient conditions and during cellular environmental stress. We revealed that the LRP8-APOE receptor-ligand system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro and helps to sustain the growth of MDA-MD-231 xenografts. We propose that inhibitors of LRP8-APOE signaling may be clinically useful therapeutic agents for triple-negative breast cancer.

Mechanism of silica -induced apoptosis in human alveolar macrophages

The mechanisms involved in the development of pulmonary silicosis have not been well defined, however most current evidence implicates a central role for alveolar macrophages in this process. We propose that the fibrotic potential of a particulate depends upon its ability to cause apoptosis in alveolar macrophage (AM). The overall goal of this study was to determine the mechanism of silica-induced apoptosis of AM. Human AM were treated with fibrogenic, poorly fibrogenic and nonfibrogenic model particulates, such as, silica, amorphous silica and titanium dioxide, respectively (equal surface area). Treatment with silica resulted in apoptosis in human AM as observed by morphology, DNA fragmentation and Cell Death ELISA assays. In contrast, amorphous silica and titanium dioxide demonstrated no significant apoptotic potential. To elucidate the possible mechanism by which silica causes apoptosis, we investigated the role of the scavenger receptor (SR) in silica-induced apoptosis. Cells were pretreated with and without SR ligand binding inhibitors, polyinosinic acid (Poly I), fucoidan and high density lipoprotein (HDL), prior to silica treatment. Pretreatment with Poly I and fucoidan resulted in significant inhibition of silica-induced apoptosis suggesting that silica-induced AM apoptosis is mediated via the SR. Further, we examined the involvement of interleukin converting enzyme (ICE) family of proteases in silica-mediated apoptosis. Silica activated ICE, Ich-1L, cpp32 beta and cleavage of PARP. Taken together, these results suggested that (1) fibrogenic particulates, such as, silica caused apoptosis of alveolar macrophages, (2) this apoptotic potential of fibrogenic particulates may be a critical factor in initiating an inflammatory response resulting in fibrosis, (3) silica-induced apoptosis of alveolar macrophages may be due to the interaction of silica particulates with the SR, and (4) silica-induced apoptosis involves the activation of the ICE family of proteases. An understanding of the molecular events involved in fibrogenic particulate-induced apoptosis may provide a useful insight into the mechanism involved in particulate-induced fibrosis. ^

Metabolic risk score indexes validation in overweight healthy people

The constellation of adverse cardiovascular disease (CVD) and metabolic risk factors, including elevated abdominal obesity, blood pressure (BP), glucose, and triglycerides (TG) and lowered high-density lipoprotein-cholesterol (HDL-C), has been termed the metabolic syndrome (MetSyn) [1]. A number of different definitions have been developed by the World Health Organization (WHO) [2], the National Cholesterol Education Program Adult Treatment Panel III (ATP III) [3], the European Group for the Study of Insulin Resistance (EGIR) [4] and, most recently, the International Diabetes Federation (IDF) [5]. Since there is no universal definition of the Metabolic Syndrome, several authors have derived different risk scores to represent the clustering of its components [6-11].

Effects of dietary restriction combined with different exercise programs or physical activity recommendations on blood lipids in overweight adults

Background and aim: Many exercise studies, although generally showing the beneficial effects of supervised aerobic, resistance or combined exercise on blood lipids, have sometimes reached equivocal conclusions. The aim of this study is to evaluate the impact of different programs that combined exercise and dietary restriction on blood lipids versus a clinical practice intervention for weight loss, in overweight adults. Methods: For this study 66 subjects participated in a supervised 22 weeks training program, composed of three sessions per week and they were randomized in three groups: strength training (S; n = 19), endurance training (E; n = 25), a combination of E and S (SE; n = 22). Eighteen subjects served as physical activity group (PA) that followed a clinical intervention consisted of physical activity recommendations. All groups followed the same dietary treatment, and blood samples were obtained for lipids measurements, at the beginning and end of the study. Results: Lipid profile improved in all groups. No significant differences for baseline and post-training values were observed between groups. In general, SE and PA decreased low-density lipoprotein cholesterol (LDL-C) values (p menor que 0.01). S decreased triglyceride levels (p menor que 0.01) and E, SE, and PA decreased total cholesterol levels (p menor que 0.05, p menor que 0.01 and p menor que 0.01, respectively). Conclusions: These results suggest that an intervention program of supervised exercise combined with diet restriction did not achieved further improvements in blood lipid profile than diet restriction and physical activity recommendations, in overweight adults. (Clinical Trials gov number: NCT01116856).

Análisis de los efectos de un programa nutricional y de ejercicio físico sobre el perfil lipídico en personas con sobrepeso y obesidad

Introducción. Las enfermedades cardiovasculares (ECV) son la principal causa de muerte en nuestro país. Entre los factores independientes más importantes para el desarrollo de ECV se encuentran en primer lugar las alteraciones del perfil lipídico como el aumento del colesterol total (TC), las lipoproteínas de baja densidad (LDL) y los triglicéridos (TG) y/o la disminución de las lipoproteínas de alta densidad (HDL). Entre las diferentes formas de abordar el problema para prevenir y tratar estas patologías se encuentra la modificación de los hábitos de vida a través de un programa combinado de dieta y ejercicio. La evidencia confirma la efectividad de la primera variable, sin embargo, en el empleo del ejercicio se encuentran discrepancias acerca de cuál es el modo más eficaz para mejorar el perfil lipídico. Objetivo. Estudiar los cambios en las variables del perfil lipídico y los índices lipoproteicos comparando cuatro tipos de intervención que combinan dieta y diferentes modos de ejercicio, así como, analizar otras variables independientes (género, edad y genotipo ApoE) que pueden tener influencia sobre estos cambios. Diseño de la investigación. Los datos analizados en esta tesis forman parte del estudio “PROgramas de Nutrición y Actividad Física para el tratamiento de la obesidad” (PRONAF). Se trata de un estudio clínico desarrollado en España entre el 2008 y el 2011. La metodología del estudio nos permite comparar cuatro tipos de intervención para la pérdida de peso y evaluar su impacto sobre el perfil lipídico. El diseño fue experimental aleatorizado donde a todos los participantes se les sometió a un programa de dieta equilibrada hipocalórica junto a uno de los tres modos de ejercicio (grupo de entrenamiento de fuerza, grupo de entrenamiento de resistencia y grupo de entrenamiento combinado de los modos anteriores; los cuales fueron igualados en volumen e intensidad) o grupo de recomendaciones de actividad física. Las principales variables analizadas en los estudios que comprende esta tesis doctoral fueron: HDL, LDL, TG y TC, los índices derivados de estas y variables de la composición corporal y del entrenamiento. Conclusiones. Los cuatro tipos de intervención mostraron ser favorables para mejorar las variables del perfil lipídico y los índices lipoproteicos, sin diferencias significativas entre ellos. Tras la intervención, los varones mostraron una respuesta más favorable en los cambios del perfil lipídico. El genotipo ApoE2 obtuvo una reducción mayor en la concentración de TG y TC que el genotipo ApoE3 y ApoE4. Por último, los índices lipoproteicos mejoraron tras un programa de pérdida de peso, obteniéndose mayores cambios en el grupo de dieta más entrenamiento aeróbico para los índices ApoB/ApoA-1, TG/HDL y LDL/ApoB. ABSTRACT Introduction. The main cause of death in our country is cardiovascular disease (CVD). The most important independent factors for the development of CVD are the lipid profile alterations: increased total cholesterol (TC), low density lipoprotein (LDL) and triglycerides (TG) and/or decreased high-density lipoprotein (HDL). Among the different approaches to prevent and treat these diseases is modifying the lifestyle combining a diet and exercise program. The evidence confirms the effectiveness of the first variable, however, there is still controversy about the most effective mode of exercise combined with diet to achieve improvements. Objective. To study changes in lipoprotein profile comparing four types of intervention combining diet with different modes of exercise, and to analyze the independent variables (gender, age, and ApoE genotype) that can influence these changes. Research design. The data analized in this thesis are part of the study Nutrition and Physical Activity Programs for Obesity Treatments (the PRONAF study according to its Spanish initials). This is a clinical research carried out in Spain between 2008 and 2011. The aim of this study was to compare four types of intervention to weight loss with diet combining exercise. The design was experimental randomized where all participants were subjected to follow a hypocaloric balanced diet along one of the three modes of exercise (strength training group, resistance training group and combined training group of the above modes, which were matched by volume and intensity) or physical activity recommendations group. The main variables under investigation in this thesis were: HDL, LDL, TG and TC, the lipoprotein ratios, body composition and training variables. Main outcomes. The four types of interventions shown to be favorable to improve the lipid profile and lipoprotein level, with no significant differences between intervention groups. After the intervention, the men showed a more favorable respond in lipid profile changes. The genotype ApoE2 obtained more positive changes in the concentration of TG and TC than ApoE3 and ApoE4 genotype. Last, the lipoprotein ratios improve after weight loss treatment with diet combined different modes exercise. Our results reflected greater changes for E group in apoB/ApoA1, TG/HDL and LDL/ApoB compared within different intervention groups.

Influence of glycerin and lecithin inclusion in the diet on liver characteristics and lipid fraction in the serum of brownegg laying hens at 55 week of age

The effects of the inclusion of raw glycerin (GLYC) and raw lecithin, in the diet (23 to 55 wk) on liver characteristics and various serum lipid fractions were studied in brown egg-laying hens at 55 wk of age. The control diets were based on corn, soybean meal, and 4% supplemental fat and contained 2,750 kcal AMEn/kg, 16.5% CP, and 0.73% digestible Lys. The diets were arranged as a 2 × 3 factorial with 2 levels of GLYC (0 and 7%) and 3 animal fat to lecithin ratios (4:0, 2:2, and 0:4%). Each treatment was replicated 8 times and the experimental unit was a cage with 10 hens. At 55 wk of age, 2 hens per cage replicate were randomly selected, weighed individually, and slaughtered by CO2 inhalation. Liver was immediately removed and weighed and the color recorded by spectrophotometry. In addition, blood samples from one bird per replicate were collected from the wing vein and the concentration of total cholesterol, low and high density lipoprotein cholesterol, and triglycerides were determined. The data were analyzed as a completely randomized design and the main effects of GLYC and lecithin content of the diet and the interactions were determined. No interactions between GLYC and lecithin content of the diets were detected for any of the variables studied. Liver characteristics and serum lipid traits were not affected by the inclusion of GLYC in the diet. The substitution of animal fat by lecithin, however, reduced the redness (a* 14.9 to 13.8) and yellowness (b* 8.60 to 7.20) values of the liver (P < 0.05) but did not affect the content of serum lipid fractions. It is concluded that the inclusion of GLYC and lecithin in the diet did not affect liver size or serum lipid fraction. However, the inclusion of lecithin reduced the a* and b* value of the liver

Low-dose expression of a human apolipoprotein E transgene in macrophages restores cholesterol efflux capacity of apolipoprotein E-deficient mouse plasma

Apolipoprotein E- (apoE) deficient (E−/−) mice develop severe hyperlipidemia and diffuse atherosclerosis. Low-dose expression of a human apoE3 transgene in macrophages of apoE-deficient mice (E−/−hTgE+/0), which results in about 5% of wild-type apoE plasma levels, did not correct hyperlipidemia but significantly reduced the extent of atherosclerotic lesions. To investigate the contribution of apoE to reverse cholesterol transport, we compared plasmas of wild-type (E+/+), E−/−, and E−/−hTgE+/0 mice for the appearance of apoE-containing lipoproteins by electrophoresis and their capacity to take up and esterify 3H-labeled cholesterol from radiolabeled fibroblasts or J774 macrophages. Wild-type plasma displayed lipoproteins containing apoE that were the size of high density lipoprotein and that had either electrophoretic α or γ mobilities. Similar particles were also present in E−/−hTgE+/0 plasma. Depending on incubation time, E−/− plasma released 48–74% less 3H-labeled cholesterol from fibroblasts than E+/+ plasma, whereas cholesterol efflux into E−/−hTgE+/0 plasma was only 11–25% lower than into E+/+ plasma. E−/−hTgE+/0 plasma also released 10% more 3H-labeled cholesterol from radiolabeled J774 macrophages than E−/− plasma. E+/+ and E−/−hTgE+/0 plasma each esterified significantly more cell-derived 3H-labeled cholesterol than E−/− plasma. Moreover, E−/− plasma accumulated much smaller proportions of fibroblast-derived 3H-labeled cholesterol in fractions with electrophoretic γ and α mobility than E+/+ and E−/−hTgE+/0 plasma. Thus, low-dose expression of apoE in macrophages nearly restored the cholesterol efflux capacity of apoE-deficient plasma through the formation of apoE-containing particles, which efficiently take up cell-derived cholesterol, and through the increase of cholesterol esterification activity. Thus, macrophage-derived apoE may protect against atherosclerosis by increasing cholesterol efflux from arterial wall cells.

Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are highly expressed during embryonic development

Apolipoprotein E (apoE) is associated with several classes of plasma lipoproteins and mediates uptake of lipoproteins through its ability to interact with specific cell surface receptors. Besides its role in cardiovascular diseases, accumulating evidence has suggested that apoE could play a role in neurodegenerative diseases, such as Alzheimer disease. In vertebrates, apoA-I is the major protein of high-density lipoprotein. ApoA-I may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway. We have isolated cDNA clones that code for apoE and apoA-I from a zebrafish embryo library. Analysis of the deduced amino acid sequences showed the presence of a region enriched in basic amino acids in zebrafish apoE similar to the lipoprotein receptor-binding region of human apoE. We demonstrated by whole-mount in situ hybridization that apoE and apoA-I genes are highly expressed in the yolk syncytial layer, an extraembryonic structure implicated in embryonic and larval nutrition. ApoE transcripts were also observed in the deep cell layer during blastula stage, in numerous ectodermal derivatives after gastrulation, and after 3 days of development in a limited number of cells both in brain and in the eyes. Our data indicate that apoE can be found in a nonmammalian vertebrate and that the duplication events, from which apoE and apoA-I genes arose, occurred before the divergence of the tetrapod and teleost ancestors. Zebrafish can be used as a simple and useful model for studying the role of apolipoproteins in embryonic and larval nutrition and of apoE in brain morphogenesis and regeneration.

The epitopes for some antiphospholipid antibodies are adducts of oxidized phospholipid and β2 glycoprotein 1 (and other proteins)

Circulating autoantibodies to phospholipids (aPLs), such as cardiolipin (CL), are found in patients with antiphospholipid antibody syndrome (APS). We recently demonstrated that many aPLs bound to CL only after it had been oxidized (OxCL), but not to a reduced CL analogue that could not undergo oxidation. We now show that the neoepitopes recognized by some aPLs consist of adducts formed between breakdown products of oxidized phospholipid and associated proteins, such as β2 glycoprotein 1 (β2GP1). Addition of human β2GP1, polylysine, native low-density lipoprotein, or apolipoprotein AI to OxCL-coated wells increased the anticardiolipin antibody (aCL) binding from APS sera that first had been diluted so that no aCL binding to OxCL could be detected. No increase in aCL binding was observed when these proteins were added to wells coated with reduced CL. The ability of β2GP1, polylysine, or low-density lipoprotein to be a “cofactor” for aCL binding to OxCL was greatly reduced when the proteins were methylated. Incubation of β2GP1 with oxidized 1-palmitoyl-2-linoleyl-[1-14C]-phosphatidylcholine (PC), but not with dipalmitoyl-[1-14C]-PC, led to formation of covalent adducts with β2GP1 recognized by APS sera. These data suggest that the reactive groups of OxCL, such as aldehydes generated during the decomposition of oxidized polyunsaturated fatty acids, form covalent adducts with β2GP1 (and other proteins) and that these are epitopes for aCLs. Knowledge that the epitopes recognized by many aPLs are adducts of oxidized phospholipid and associated proteins, including β2GP1, may give new insights into the pathogenic events underlying the clinical manifestations of APS.

Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Substantial narrowing of the Niemann–Pick C candidate interval by yeast artificial chromosome complementation

Niemann–Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11–12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

The solution structure of the N-terminal domain of α2-macroglobulin receptor-associated protein

The three-dimensional structure of the N-terminal domain (residues 18–112) of α2-macroglobulin receptor-associated protein (RAP) has been determined by NMR spectroscopy. The structure consists of three helices composed of residues 23–34, 39–65, and 73–88. The three helices are arranged in an up-down-up antiparallel topology. The C-terminal 20 residues were shown not to be in a well defined conformation. A structural model for the binding of RAP to the family of low-density lipoprotein receptors is proposed. It defines a role in binding for both the unordered C terminus and the structural scaffold of the core structure. Pathogenic epitopes for the rat disease Heymann nephritis, an experimental model of human membranous glomerulonephritis, have been identified in RAP and in the large endocytic receptor gp330/megalin. Here we provide the three-dimensional structure of the pathogenic epitope in RAP. The amino acid residues known to form the epitope are in a helix–loop–helix conformation, and from the structure it is possible to rationalize the published results obtained from studies of fragments of the N-terminal domain.