RESUMEN
BACKGROUND: There are limited data available on the longitudinal relationship between candy consumption by children on weight and other cardiovascular risk factors (CVRF) in young adults. The present study investigated whether candy consumption in children was predictive of weight and CVRF in young adults. METHODS: A longitudinal sample of children 10 years (n = 355; 61% females; 71% European-Americans, 29% African-Americans) who participated in cross-sectional surveys from 1973 to 1984 (baseline) and in one of two surveys (follow-ups) as young adults [19-38 years; mean (SD) = 23.6 (2.6) years] in Bogalusa, LA, were studied. Dietary data were collected using 24-h dietary recalls at baseline and at one follow-up survey; a food frequency questionnaire was used in the other follow-up survey. Candy consumers were those consuming any amount of candy. Candy consumption was calculated (g day(-1) ) from baseline 24-h dietary recalls, and was used as a covariate in the adjusted linear mixed models. Dependent variables included body mass index (BMI) and CVRF measured in young adults. RESULTS: At baseline, 92% of children reported consuming candy [46 (45) g day(-1)]; the percentage decreased to 67% [20 (30) g day(-1)] at follow-up. No longitudinal relationship was shown between baseline candy consumption and BMI or CVRF in young adults, suggesting that candy consumption was not predictive of health risks later in life. CONCLUSIONS: The consumption of nutrient rich foods consistent with dietary recommendations is important, although modest amounts of candy can be added to the diet without potential adverse long-term consequences to weight or CVRF. Additional studies are needed to confirm these results.
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Índice de Masa Corporal , Dulces , Enfermedades Cardiovasculares/etiología , Dieta , Conducta Alimentaria , Obesidad/etiología , Adiposidad , Adulto , Negro o Afroamericano , Dulces/efectos adversos , Niño , Estudios Transversales , Dieta/efectos adversos , Ingestión de Energía , Femenino , Humanos , Estudios Longitudinales , Louisiana , Masculino , Encuestas y Cuestionarios , Población Blanca , Adulto JovenRESUMEN
OBJECTIVE: To investigate possible age-related changes in associations between polymorphisms in the fat mass and obesity-associated (FTO) gene and higher body mass index (BMI). DESIGN AND SUBJECTS: Multilevel mixed regression models were used to examine associations between four FTO variants and longitudinal BMI profiles in non-Hispanic white and African American children and adolescents 8-17 years of age from two different longitudinal cohort studies, the Bogalusa Heart Study (BHS) and Project HeartBeat! (PHB). In the BHS, there were 1551 examinations of 478 African Americans and 3210 examinations of 1081 non-Hispanic whites; in PHB, there were 971 examinations of 131 African Americans and 4458 examinations of 505 non-Hispanic whites. RESULTS: In African Americans, no significant FTO associations with BMI were found. In non-Hispanic whites, linkage disequilibrium among all four variants made haplotype analysis superfluous, so we focused on the single-nucleotide polymorphism, rs9939609. In longitudinal multilevel models, the A/A genotype of rs9939609 was associated with higher BMI in non-Hispanic whites in both cohorts at all ages. A significant age-by-genotype interaction found only in the BHS cohort predicted that in those with the A/A genotype, BMI would be â¼0.7 kg m(-2) higher at age 8 and â¼1.6 kg m(-2) higher at age 17 than in those with A/T or T/T genotypes. The design of PHB limited follow-up of any single individual to 4 years, and may have reduced the ability to detect any age-by-genotype interaction in this cohort. CONCLUSIONS: The A/A genotype of rs9939609 in the FTO gene is associated with higher longitudinal BMI profiles in non-Hispanic whites from two different cohorts. The association may change with age, with the A/A genotype being associated with a larger BMI difference in late adolescence than in childhood, though this was observed only in the BHS cohort and requires verification.
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Aterosclerosis/genética , Negro o Afroamericano/genética , Resistencia a la Insulina/genética , Obesidad/genética , Polimorfismo de Nucleótido Simple , Proteínas/genética , Población Blanca/genética , Adolescente , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Aterosclerosis/epidemiología , Aterosclerosis/etnología , Niño , Estudios de Cohortes , Femenino , Humanos , Resistencia a la Insulina/etnología , Desequilibrio de Ligamiento , Estudios Longitudinales , Louisiana/epidemiología , Masculino , Análisis Multinivel , Obesidad/epidemiología , Obesidad/etnología , ProhibitinasRESUMEN
Children from an entire biracial geographical population were examined for blood pressure. A sample of 278 children, stratified by diastolic blood pressure, was reexamined 1 to 2 years later. Dopamine beta-hydroxylase, renin activity, and resting heart rate were observed in black and white children. In the group with high blood pressure, whites had higher heart rates and greater renin activity than blacks. Dopamine beta-hydroxylase concentrations in blacks were lower than in whites over the entire spectrum of blood pressure levels. High blood pressure seems to have a different metabolic background in the two races which may influence the early natural history of essential hypertension. Therefore, the rationale of prevention, and possibly treatment, of early hypertension in blacks and whites may differ.
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Población Negra , Presión Sanguínea , Dopamina beta-Hidroxilasa/sangre , Renina/sangre , Población Blanca , Adolescente , Niño , Preescolar , Creatinina/orina , Frecuencia Cardíaca , Humanos , Factores Sexuales , Sodio/orinaRESUMEN
OBJECTIVE: Although obese children are at increased risk for coronary heart disease in later life, it is not clear if the association results from the persistence of childhood obesity into adulthood. We examined the relation of both childhood and adult levels of body mass index (BMI, kg m(-2)) to carotid intima-media thickness (IMT) measured at the (mean) age of 36 years. DESIGN AND SUBJECTS: Prior to the determination of adult IMT, the 1142 participants had been examined 7 (mean) times in the Bogalusa Heart Study. MEASUREMENTS: In addition to BMI, levels of lipids, lipoproteins and blood pressure were measured at each examination. Cumulative levels of each risk factor were based on the areas under the individual growth curves calculated using multilevel models for repeated (BMI) measurements. We then examined the relation of these cumulative levels to adult IMT. RESULTS: Carotid IMT was associated with cumulative levels of BMI in both childhood and adulthood (P<0.001 for each association). Furthermore, the association between childhood BMI and adult IMT persisted, but was reduced, after controlling for adult BMI. Although childhood levels of lipids, lipoproteins and blood pressure were also associated with adult IMT, these associations were not independent of adult levels of these risk factors. CONCLUSIONS: These results emphasize the adverse effects of elevated childhood BMI levels. In addition to the strong tracking of BMI levels from childhood to adulthood, there appears to be a modest, independent effect of childhood BMI on adult IMT. The prevention of childhood obesity should be emphasized.
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Aterosclerosis/etiología , Índice de Masa Corporal , Arterias Carótidas/patología , Obesidad/complicaciones , Túnica Media/patología , Adulto , Aterosclerosis/patología , Arterias Carótidas/diagnóstico por imagen , Niño , Preescolar , Femenino , Humanos , Lípidos/sangre , Masculino , Obesidad/patología , Obesidad/prevención & control , Túnica Media/diagnóstico por imagen , UltrasonografíaRESUMEN
We studied the metabolism of lipoprotein-proteoglycan complexes by macrophage-derived foam cells. Foam cells were isolated from atherosclerotic rabbit aortas. ApoB-lipoprotein-proteoglycan complex was isolated from human aorta fibrous plaque lesions and LDL-proteoglycan complex was formed in vitro. Both in vitro and in vivo complexes stimulated cholesteryl ester synthesis in foam cells by a dose-dependent, saturable process that resulted in the intracellular accumulation of cholesteryl ester. Stimulation of cholesteryl ester synthesis was linear with time over a 32-h period. Polyinosinic acid inhibited the stimulation of cholesteryl ester synthesis by the complexes by 32-37%, whereas cytochalasin D only produced a 6-16% inhibition. Foam cells degraded 125I-LDL-proteoglycan complex and 125I-acetyl LDL in a saturable, dose-dependent manner. Excess unlabeled acetyl-LDL inhibited the degradation of 125I-LDL-proteoglycan complex by 52%, while LDL had no effect. Similarly, excess unlabeled complex suppressed the degradation of 125I-acetyl-LDL by 48%. Foam cells degraded 125I-methyl-LDL-proteoglycan complex to the same extent as 125I-LDL-proteoglycan complex. These results show that foam cells from atherosclerotic lesions metabolize lipoprotein-proteoglycan complexes predominantly via receptor-mediated endocytosis and consequently continue to accumulate intracellular cholesteryl ester.
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Aorta/metabolismo , Arteriosclerosis/metabolismo , Ésteres del Colesterol/metabolismo , Células Espumosas/metabolismo , Arteria Ilíaca/metabolismo , Lipoproteínas LDL/farmacología , Músculo Liso Vascular/metabolismo , Proteoglicanos/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/patología , Arteriosclerosis/patología , Colesterol/metabolismo , Colesterol en la Dieta , Sulfatos de Condroitina/farmacología , Citocalasina D/farmacología , Dermatán Sulfato/farmacología , Dieta Aterogénica , Células Espumosas/efectos de los fármacos , Células Espumosas/patología , Humanos , Arteria Ilíaca/efectos de los fármacos , Arteria Ilíaca/patología , Cinética , Lipoproteínas LDL/sangre , Lipoproteínas LDL/aislamiento & purificación , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Poli I/farmacología , ConejosRESUMEN
BACKGROUND: The determinants of differences in blood pressure that emerge in adolescence between black Americans of predominantly African descent and white Americans of predominantly European descent are unknown. One hypothesis is related to intrauterine and early childhood growth. The role of early blood pressure itself is also unclear. We tested whether differences in birth weight and in carefully standardized subsequent measures of weight, height, and blood pressure from 0 to 4 or 5 years were related to black/white differences in blood pressure in adolescence. METHODS AND RESULTS: Two Bogalusa cohorts who had complete follow-up data on birth weights and early childhood and adolescent anthropometric and blood pressure measures were pooled. One hundred eighty-five children (48 black and 47 white boys and 41 black and 49 white girls) were followed up and studied after 15 to 17 years. Birth weights were a mean 443 and 282 g lower in black boys and girls, respectively, than in whites (P<0.001). Blood pressures in adolescence were 3.4/1.9 and 1.7/0.6 mm Hg higher, respectively, and tracked from early childhood. In regression analyses, birth weight accounted for the ethnic difference in adolescent blood pressure, which was also independently predicted, in decreasing impact order, by adolescent height, adolescent body mass index, and systolic blood pressure at 4 to 5 years and inversely by growth from 0 to 4 to 5 years. CONCLUSIONS: If these results can be replicated in larger and independent samples, they suggest that efforts to improve intrauterine growth in black infants as well as lessen weight gain in adolescence might substantially reduce excess high blood pressure/hypertension in this ethnic group.
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Peso al Nacer/fisiología , Presión Sanguínea/fisiología , Tamaño Corporal/fisiología , Crecimiento/fisiología , Hipertensión/etnología , Adolescente , Población Negra , Índice de Masa Corporal , Preescolar , Desarrollo Fetal/fisiología , Humanos , Hipertensión/etiología , Lactante , Recién Nacido , Modelos Logísticos , Población BlancaRESUMEN
OBJECTIVE: To understand tracking of overweight status from childhood to young adulthood in a biracial sample. DESIGN: A longitudinal sample was created from cross-sectional surveys at two time points, childhood (baseline) and young adulthood (follow-up). SETTING: Bogalusa Heart Study, Louisiana, United States of America. SUBJECTS: A total of 841 young adults, 19-35 years (68% Euro-Americans (EA), 32% African-Americans (AA)) were studied. The same subjects had also participated in one of the five cross-sectional surveys at childhood (9-11 years). METHODS: Body mass index (BMI) was used to determine overweight status as per the Centers for Disease Control and Prevention standards. Change in the BMI status from childhood to young adulthood was used to group the participants into the following categories: normal weight to normal weight (NW-NW); normal weight to overweight (NW-OW); overweight to normal weight (OW-NW); and overweight to overweight (OW-OW). Tracking of overweight was defined by (1) correlations between baseline and follow-up BMI, (2) Cohen's kappa concordance test to determine the strength of tracking in BMI quartiles and (3) the percentage of individuals who remained in the same overweight status group from baseline to follow-up. RESULTS: From baseline to follow-up, the percentage of participants who were overweight increased from 24.7 to 57.7%. A total of 35.2% of the children shifted from normal weight in childhood to overweight in young adulthood (P < 0.0005). Baseline BMI was positively correlated with follow-up BMI (r = 0.66, P < 0.0005). A total of 61.9% of the participants in the highest BMI quartile in childhood remained in the highest BMI quartile in young adulthood. The strength of tracking in BMI quartiles was 27% for EA men (P < 0.0005), 23% for EA women (P < 0.0005), 27% for AA men (P<0.0005) and 35% for AA women (P < 0.0005). A total of 53.7% of the EA women remained in the NW-NW category and 31.2% of the AA women remained in the OW-OW category. The percentage tracking (NW-NW and OW-OW) was 72.8% in EA women, 59.6% in AA men, 59.5% in AA women and 48.8% in EA men (P < 0.0001). CONCLUSION: Childhood overweight tracked into young adulthood in this sample and the tracking of NW-NW and OW-OW was the most prominent among the EA women.
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Negro o Afroamericano/estadística & datos numéricos , Índice de Masa Corporal , Obesidad/epidemiología , Población Blanca/estadística & datos numéricos , Adulto , Peso Corporal/fisiología , Niño , Estudios de Cohortes , Estudios Transversales , Femenino , Estudios de Seguimiento , Humanos , Louisiana/epidemiología , Masculino , Obesidad/etnología , Valor Predictivo de las Pruebas , Factores SexualesRESUMEN
A chondroitin sulfate-dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by beta-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.
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Aorta/análisis , Sulfatos de Condroitina/aislamiento & purificación , Condroitín/análogos & derivados , Dermatán Sulfato/aislamiento & purificación , Glicosaminoglicanos/aislamiento & purificación , Proteoglicanos/aislamiento & purificación , Animales , Bovinos , Centrifugación por Gradiente de Densidad , Condroitín Liasas , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Peso Molecular , Fragmentos de Péptidos/análisis , TripsinaRESUMEN
We studied the effect of bovine endothelial cell-conditioned medium on proteoglycan synthesis by bovine aorta smooth muscle cells. Confluent cultures were incubated with [35S]sulfate, [3H]glucosamine or [3H]serine in medium alone (control), or medium that had been conditioned on confluent endothelial cells. Metabolically labelled proteoglycans secreted into the culture medium and associated with the cell layer were quantified. During a 24 h incubation, endothelial cell-conditioned medium increased [35S]sulfate and [3H]glucosamine incorporation into medium and cell-layer proteoglycans by 59% and 95%, respectively, above controls. [3H]Serine incorporation into proteoglycan core protein was increased by 150%. The effect of endothelial cell-conditioned medium on [35S]sulfate incorporation was concentration dependent. The stimulatory effects of the conditioned medium were abolished by cycloheximide and actinomycin D, inhibitors of protein synthesis and transcription, respectively. Endothelial cell-conditioned medium caused no significant change in the degradation or secretion of proteoglycans, indicating that the increase in proteoglycans was due to increased de novo synthesis. TGF-beta neutralizing antibody inhibited 22% of the stimulatory effect of the conditioned medium, suggesting that part of the stimulation was mediated by TGF-beta. Ion-exchange chromatography of [35S]proteoglycans in the culture medium of smooth muscle cells yielded two major peaks at 0.52 and 0.57 M NaCl in both control and experimental cultures. In both cases the second peak, which represented approx. 80% of the total radioactivity, contained isomeric chondroitin sulfate proteoglycan with chondroitin sulfate and dermatan sulfate accounting for 90% and 10% of the isomers, respectively. The isomeric chondroitin sulfate proteoglycan was fractionated by hydrodynamic size on Sepharose CL-4B, resulting in three fractions (A, B and C). Analytical column chromatography of fractions A and B on Sepharose CL-2B demonstrated that proteoglycans from cultures incubated with endothelial cell-conditioned medium were larger in size than those from control cultures (M(r) fraction A, 1700,000, compared with 1200,000 M(r); fraction B, 540,000, compared with 390,000). The molecular weights of the core proteins were unchanged. The larger size of proteoglycan A in cultures exposed to endothelial cell-conditioned medium was due to an increase in both the glycosaminoglycan chain number (29 compared to 25) and molecular mass (M(r) 52,000, compared to 40,000). The hydrodynamic size of the glycosaminoglycans in proteoglycan B of control and experimental cultures was identical (M(r) 40,000). Therefore, the increase in the molecular mass of this proteoglycan was attributable to an increase in glycosaminoglycan chain number (12 compared to 9).(ABSTRACT TRUNCATED AT 400 WORDS)
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Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Medios de Cultivo/farmacología , Músculo Liso Vascular/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Bovinos , Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Cicloheximida , Dactinomicina , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Enzimas , Glucosamina/metabolismo , Calor , Serina/metabolismo , Sulfatos/metabolismo , Factores de TiempoRESUMEN
A heparan sulfate proteoglycan from bovine lung gas-exchange tissue was isolated by extraction of the tissue with 4.0 M guanidine HCl in the presence of multiple protein inhibitors. The proteoglycan was purified by precipitation with cetylpyridinium chloride in 0.5 M KCl followed by CsCl isopycnic centrifugation (po = 1.45) in 4.0 M guanidine/HCl. Further purification was achieved by gel filtration on Sepharose CL-2B and by chromatography in DEAE-Sepharose CL-6B column. The proteoglycan had 14.9% protein and 22.4% uronate. Heparan sulfate chains from the proteoglycan were isolated after beta-elimination. Fractionation of heparan sulfate chains was achieved on Dowex-1 Cl- column, eluting with a stepwise increase in the concentration of NaCl, 1.0 to 2.0 M with 0.2 M increments. Of the total heparan sulfate recovered from the column, about 10% eluted by 1.2 M NaCl, 68% by 1.4 M NaCl, 18% by 1.6 M NaCl and 4% by 1.8 M NaCl. The fractions varied in their total and N-sulfate ester contents and iduronic acid to glucuronic acid ratios. The fraction that eluted from the Dowex-1 Cl- column at 1.6 M NaCl had the highest molecular weight, 37000, and the fraction that eluted at 1.8 M NaCl had the lowest molecular weight, 12000, as determined by gel filtration method, and the greatest sulfate content. The core protein, obtained by digestion of proteoglycan by heparan sulfate lyase, showed mostly a single band in SDS-polyacrylamide gel electrophoresis. The observations indicate a heterogeneity of the composition of heparan sulfate chains in the proteoglycan. This heterogeneity likely contributes to variations in biologic properties of different heparan sulfate proteoglycan preparations.
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Proteoglicanos Tipo Condroitín Sulfato/análisis , Glicosaminoglicanos/análisis , Heparitina Sulfato/análisis , Pulmón/análisis , Proteoglicanos/análisis , Animales , Bovinos , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Proteoglicanos de Heparán SulfatoRESUMEN
A high-affinity heparin subfraction accounting for 8% of whole heparin from bovine lung was isolated by low-density lipoprotein (LDL)-affinity chromatography. When compared to whole heparin, the high-affinity subfraction was relatively higher in molecular weight (11,000 vs. 17,000) and contained more iduronyl sulfate as hexuronic acid (76% vs. 86%), N-sulfate ester (0.75 vs. 0.96 mol/mol hexosamine), and O-sulfate ester (1.51 vs. 1.68 mol/mol hexosamine). Although both heparin preparations formed insoluble complexes with LDL quantitatively in the presence of 30 mM Ca2+, the concentrations of NaCl required for 50% reduction in maximal insoluble complex formation was markedly higher with high-affinity subfraction (0.55 M vs. 0.04 M). When compared to complex of 125I-LDL and whole heparin (H-125I-LDL), complex of 125I-LDL and high-affinity heparin subfraction (HAH-125I-LDL) produced marked increase in the degradation of lipoproteins by macrophages (7-fold vs. 1.4-fold over native LDL, after 5 h incubation) as well as cellular cholesteryl ester synthesis (16.7-fold vs. 2.2-fold over native LDL, after 18 h incubation) and content (36-fold vs. 2.7-fold over native LDL, after 48 h incubation). After a 5 h incubation, macrophages accumulated 2.3-fold more cell-associated radioactivity from HAH-125I-LDL complex than from [125I]acetyl-LDL. While unlabeled HAH-LDL complex produced a dose-dependent inhibition of the degradation of labeled complex, native unlabeled LDL did not elicit any effect even at a 20-fold excess concentration. Unlabeled particulate LDL aggregate competed for 33% of degradation of labeled complex; however, cytochalasin D, known inhibitor of phagocytosis, did not effectively inhibit the degradation of labeled complex. Unlabeled acetyl-LDL produced a partial (33%) inhibition of the degradation of labeled complex. These results indicate that (1) the interaction of high-affinity heparin subfraction with LDL leads to scavenger receptor mediated endocytosis of the lipoprotein, and stimulation of cholesteryl ester synthesis and accumulation in the macrophages; and (2) with respect to macrophage recognition and uptake, HAH-LDL complex was similar but not identical to acetyl-LDL. These observations may have implications for atherogenesis, because both mast cells and endothelial cells can synthesize heparin in the arterial wall.
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Ésteres del Colesterol/metabolismo , Heparina/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Animales , Cromatografía de Afinidad , Femenino , Ligandos , Ratones , Concentración Osmolar , Cavidad Peritoneal/citologíaRESUMEN
Earlier, we (Vijayagopal, P. et al. (1988) Biochim. Biophys. Acta 960, 210) showed that mouse peritoneal macrophages metabolize low density lipoprotein (LDL)-proteoglycan complex by a receptor pathway distinct from the acetyl-LDL receptor. Further studies were conducted to probe further into the mechanism of LDL-proteoglycan complex uptake by macrophages. Both 125I-methyl-LDL-proteoglycan complex and 125I-LDL-proteoglycan complex were taken up and degraded by the cells to the same extent. Similarly, the ability of these ligands to stimulate cholesteryl ester synthesis was also indistinguishable. These results rule out the possibility of apoB,E receptor involvement in the uptake of LDL-proteoglycan complex in macrophages. Sodium fluoride, cytochalasin D and aggregated LDL inhibited degradation of the complex by 24%, 26% and 28%, respectively, indicating that phagocytosis is only a minor pathway for the uptake. Both binding and degradation of the complex were not inhibited by excess hyaluronic acid suggesting that ligand recognition was not through hyaluronic acid binding sites. As compared to acetyl-LDL, the cellular degradation of LDL-proteoglycan complex was retarded. Macrophages exhibited a rapid stimulation of [3H]inositol trisphosphate (IP3) release and diacylglycerol production when incubated with LDL-proteoglycan complex. Furthermore, pertussis toxin produced a 62% inhibition of LDL-proteoglycan complex mediated IP3 release, suggesting that LDL-proteoglycan complex metabolism in macrophages is dependent upon the G-protein coupled signal transduction mechanism. These results show that receptor mediated endocytosis plays a major role in the metabolism of LDL-proteoglycan complex in macrophages.
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Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Proteoglicanos/metabolismo , Animales , Ésteres del Colesterol/metabolismo , Citocalasina D/farmacología , Diglicéridos/metabolismo , Endocitosis/fisiología , Femenino , Ácido Hialurónico/farmacología , Fosfatos de Inositol/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Cavidad Peritoneal/citología , Toxina del Pertussis , Ensayo de Unión Radioligante , Fluoruro de Sodio/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Proteoglycan aggregates were isolated from bovine aorta by extraction with 0.5 M guanidine hydrochloride in the presence of proteinase inhibitors and purified by isopycnic CsCl centrifugation. The bottom two-fifths (A1) of the gradient contained 30% of proteoglycans in the aggregated form. The aggregate had 14.8% protein and 20.4% hexuronic acid with hyaluronic acid, dermatan sulfate and chondroitin sulfates in a proportion of 18:18:69. A link protein-containing fraction was isolated from the bottom two-fifths by dissociative CsCl isopycnic centrifugation. The link protein that floated to the top one-fifth of the gradient was purified by chromatography on Sephadex G-200 in the presence of 4 M guanidine hydrochloride. It moved as a single band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 49 000. The amino acid composition of link protein resembled that of link protein from cartilage, but was strikingly different from that of the protein core of the proteoglycan monomer. The neutral sugar content of link protein was 3.5% of dry weight. Galactose, mannose and fucose constituted 21, 62 and 16%, respectively of the total neutral sugars. In aggregation studies the link protein was found to interact with both proteoglycan monomer and hyaluronic acid. Oligosaccharides derived from hyaluronic acid decreased the viscosity of link protein-free aggregates of proteoglycan and hyaluronic acid but not of link-stabilized aggregates, demonstrating that the link protein increases the stability of proteoglycan aggregates.
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Aorta/análisis , Proteínas de la Matriz Extracelular , Proteínas/aislamiento & purificación , Proteoglicanos/aislamiento & purificación , Animales , Bovinos , Ácido Hialurónico , Conformación Proteica , Solventes , ViscosidadRESUMEN
We studied the factors regulating the metabolism of low-density lipoprotein (LDL)-proteoglycan complex, LDL and acetyl-LDL in mouse peritoneal macrophages. Macrophage conditioned medium stimulated the degradation of LDL-proteoglycan complex and acetyl-LDL in a dose-dependent manner and enhanced cholesteryl ester synthesis mediated by these ligands. The conditioned medium had no such effect in a cell-free system. The conditioned medium enhanced the degradation of both the LDL and proteoglycan components of the complex. The degradation of LDL was not affected by the conditioned medium. The active factor in the conditioned medium was labile to boiling, suggesting that it may be protein in nature. The conditioned medium also lost its stimulatory activity after dialysis through a membrane with an exclusion limit of 25,000 daltons, suggesting the involvement of cytokines and/or other growth factors. Macrophage activation was accompanied by a 2-3-fold increase in the degradation of LDL-proteoglycan complex and acetyl-LDL as compared to the degradation of these ligands in resident macrophages; however, this had no effect on LDL degradation. The degradation of all three ligands increased markedly with decreasing cell density. Preincubation of macrophages for 48 h with increasing concentrations of fetal bovine serum produced a substantial increase in the subsequent degradation of LDL-proteoglycan complex and acetyl-LDL, while it had very little effect on the degradation of LDL. The active factor in serum was destroyed by boiling, suggesting that it may be a protein. These results show that the scavenger receptor, mediating the uptake and degradation of LDL-proteoglycan complex and acetyl-LDL and LDL receptor are regulated differently in mouse peritoneal macrophages.
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Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Proteoglicanos/metabolismo , Animales , Sangre , Femenino , Humanos , Ratones , Cavidad Peritoneal/citologíaRESUMEN
The characteristics of an arterial wall chondroitin sulfate proteoglycan (CS-PG) subfraction that binds avidly to low-density lipoproteins (LDL) was studied. A large CS-PG was extracted from bovine aorta intima-media under dissociative conditions, purified by density-gradient centrifugation and gel filtration chromatography, and further subfractionated by affinity chromatography on LDL-agarose. A proteoglycan subfraction, representing 25% of the CS-PG, showed an elution profile (with dissociation from LDL-agarose occurring between 0.5 and 1.0 M NaCl) corresponding to that of heparin, heretofore considered to be the most strongly binding glycosaminoglycan with LDL. The proteoglycan subfraction which migrated as a single band on composite agarose-polyacrylamide gel electrophoresis contained chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in a proportion of 70:22:8. The core protein of the proteoglycan had an apparent molecular weight of 245,000, and contained approx. 33 glycosaminoglycan chains with an average molecular weight of 32,000. The CS-PG subfraction, like heparin, formed insoluble complexes in the presence of 30 mM Ca2+. Complexing of LDL with proteoglycan resulted in two classes of interactions with 0.1 and 0.3 proteoglycan monomer bound per LDL particle characterized by an apparent Kd of 4 and 21 nM, respectively. This indicates that multiple LDL particles bind to single proteoglycan monomers even at saturation. In contrast, LDL-heparin interactions showed a major component characterized by an apparent Kd of 151 nM and a Bmax of 9 heparin molecules per LDL particle. The occurrence of a potent LDL-binding proteoglycan subfraction within the family of arterial CS-PG may be of importance in terms of lipid accumulation in atherogenesis.
Asunto(s)
Aorta/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Lipoproteínas LDL/metabolismo , Proteoglicanos/metabolismo , Aminoácidos/análisis , Animales , Bovinos , Centrifugación por Gradiente de Densidad , Condroitín Liasas/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Sulfatos de Condroitina/análisis , Cromatografía , Dermatán Sulfato/análisis , Electroforesis en Gel de Poliacrilamida , Heparina/metabolismo , Peso MolecularRESUMEN
The biologic properties of two major proteoglycans of bovine aorta, heparan sulfate proteoglycan and chondroitin sulfate-dermatan sulfate proteoglycan were compared. The heparan sulfate proteoglycan was isolated either by elastase digestion or by 4.0 M guanidine hydrochloride extraction, of aorta tissue, fractionated by CsCl isopycnic centrifugation and purified by chondroitinase ABC treatment. The first method resulted in considerably greater yield (about 70% of the total heparan sulfate proteoglycan of the tissue) than the second procedure (12% of total). The chondroitin sulfate-dermatan sulfate proteoglycan was obtained by 4.0 M guanidine-HCl extraction of aorta tissue followed by CsCl isopycnic centrifugation. The chemical composition of both heparan sulfate proteoglycan preparations was similar. Unlike the chondroitin sulfate-dermatan sulfate proteoglycan, which eluted in the void volume of Sepharose CL-6B column, the heparan sulfate proteoglycan preparations were each resolved into a high molecular weight fraction (kav = 0.18 and 0.13) and a low molecular weight fraction (kav = 0.47 and 0.36). The heparan sulfate proteoglycan preparations exhibited significantly more potent anticoagulant and platelet aggregation inhibitory activities than the chondroitin sulfate-dermatan sulfate proteoglycan. The protein core of the proteoglycan molecules did not seem to be essential for their hemostatic properties. The complex forming ability of the heparan sulfate proteoglycan with serum low density lipoproteins (LDL) was much less than that of chondroitin sulfate-dermatan sulfate proteoglycan in the presence and absence of Ca2+. Interaction between heparan sulfate proteoglycan and LDL was also much more sensitive to changes in the ionic strength of the medium than that of chondroitin sulfate-dermatan sulfate proteoglycan and the lipoprotein. Since the total sulfate content of both proteoglycans is almost similar, the smaller molecular size and hence the lower overall charge density of the heparan sulfate proteoglycan appears to be partly responsible for its low affinity for LDL. The differences in biologic properties of the two proteoglycans might have implications in the pathophysiology of cardiovascular diseases.
Asunto(s)
Anticoagulantes , Aorta Torácica/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Glicosaminoglicanos/metabolismo , Hemostasis/efectos de los fármacos , Heparitina Sulfato/metabolismo , Lipoproteínas LDL/sangre , Proteoglicanos/metabolismo , Animales , Bovinos , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Proteoglicanos Tipo Condroitín Sulfato/farmacología , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/aislamiento & purificación , Heparitina Sulfato/farmacología , Humanos , Lipoproteínas LDL/fisiología , Elastasa Pancreática/metabolismo , Tiempo de Tromboplastina Parcial , Agregación Plaquetaria/efectos de los fármacos , Tiempo de TrombinaRESUMEN
Earlier, we (Vijayagopal, P., et al. (1985) Biochim. Biophys. Acta 837-251) have shown that complexes of plasma low-density lipoproteins (LDL) and arterial chondroitin sulfate-dermatan sulfate proteoglycan aggregate promote LDL degradation and cholesteryl ester accumulation in mouse peritoneal macrophages. Further studies were conducted to determine whether LDL-proteoglycan complex is metabolized by a receptor-mediated process. Native proteoglycan aggregate was isolated from bovine aorta by associative CsCl isopycnic centrifugation. Complex of 125I-labeled LDL and proteoglycan aggregate formed in the presence of 30 mM Ca2+ was incubated with macrophages, and the binding at 4 degrees C and degradation at 37 degrees C of 125I-labeled LDL in the complex was monitored. Both binding and degradation of the complex were specific and saturable, suggesting that the processes are receptor mediated. The Kd for binding was 23 micrograms LDL protein per ml in the complex. Degradation of 125I-labeled LDL-proteoglycan complex was not suppressed by preincubation of macrophages with excess unlabeled complex, suggesting that the receptor for the complex is not subject to down regulation. Both binding and degradation of the complex and the resultant stimulation of cholesteryl ester synthesis were inhibited by limited treatment of cells with low doses of trypsin and pronase, indicating that the binding sites are protein or glycoprotein in nature. Binding was not inhibited by an excess of native LDL and beta-VLDL and exhibited only partial competition by excess unlabeled acetyl-LDL; however, polyinosinic acid, fucoidin and dextran sulfate, known inhibitors of acetyl-LDL binding and degradation in macrophages, did not affect LDL-proteoglycan complex binding and degradation. Similarly, excess unlabeled LDL-proteoglycan complex produced only partial inhibition of the binding and degradation of 125I-labeled acetyl-LDL by macrophages, suggesting that the binding sites for acetyl-LDL and LDL-proteoglycan complex are probably not identical. These studies provide evidence for a receptor-mediated pathway for the metabolism of LDL-proteoglycan complex in macrophages.
Asunto(s)
Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacología , Macrófagos/metabolismo , Proteoglicanos/metabolismo , Animales , Frío , Humanos , Cinética , Ratones , Poli I/farmacología , Polisacáridos/farmacología , Pronasa/metabolismo , Factores de Tiempo , Tripsina/metabolismoRESUMEN
The synthesis of proteoglycans by aorta explants from rabbits with diet-induced atherosclerosis and controls was studied by 35S-incorporation. Proteoglycans were isolated under dissociative conditions from incubation medium and from arterial explants. Additionally, the tissue proteoglycans that were not extracted by 4 M guanidine-HCl were solubilized by digestion of the tissue by elastase in the presence of proteinase inhibitors. The residual tissue was hydrolyzed by papain and glycosaminoglycans were isolated. The atherosclerotic aorta tissue incorporated twice the amount of 35S into proteoglycans than observed for controls; in both groups about 70% of the label incorporated into the tissue was noted in the proteoglycans extracted by guanidine-HC;, while about 30% of the total 35S-labeled proteoglycans synthesized by the explants were found in the media. Atherosclerotic tissue incorporated 35S predominantly into chondroitin sulfate proteoglycans when compared to control tissue. The chondroitinase ABC-digestable proteoglycans that were extracted by guanidine-HCl from atherosclerotic tissues were of larger molecular size than those from control tissue, but the core proteins from these preparations were similar. The heparan sulfate proteoglycan that was obtained by dissociative extraction from atherosclerotic tissue had greater amounts of N-acetyl and lesser amounts of N-sulfate ester groups than the preparation from control tissue. Digestion of the tissue by elastase yielded heparan sulfate proteoglycan as the major constituent in both groups, although atherosclerotic tissue contained relatively small amounts of this proteoglycan. The residual tissue from both groups contained chondroitin sulfate and heparan sulfate as the major glycosaminoglycans with the latter showing a decrease with atherosclerosis. Atherosclerotic tissue secreted into the medium about two-fold more 35S-labeled proteoglycans with larger molecular size than control tissue; proteoglycans of the heparan sulfate and chondroitin sulfate types were the major constituents in the culture medium of both tissues. Thus, proteoglycans undergo both quantitative and qualitative changes in atherosclerosis, reflecting the enhanced smooth muscle cell activity. These changes are potentially important in modulating lipoprotein binding and hemostatic properties, as well as fibrillogenesis of the arterial wall.
Asunto(s)
Aorta/metabolismo , Arteriosclerosis/metabolismo , Proteoglicanos/biosíntesis , Animales , Cromatografía en Gel , Dieta Aterogénica , Glicosaminoglicanos/biosíntesis , Glicosaminoglicanos/aislamiento & purificación , Guanidina , Guanidinas , Masculino , Técnicas de Cultivo de Órganos , Elastasa Pancreática , Proteoglicanos/aislamiento & purificación , Conejos , Sulfatos/metabolismo , Radioisótopos de AzufreRESUMEN
We studied the effect of complexes of low-density lipoproteins (LDL) and different proteoglycan preparations from bovine aorta on LDL degradation and cholesteryl ester accumulation in mouse peritoneal macrophages. Native proteoglycan aggregate containing proteoglycan monomers, hyaluronic acid and link protein was isolated by associative extraction of aortic tissue, while proteoglycan monomer was obtained by dissociative isopycnic centrifugation of the native proteoglycan aggregate. In vitro proteoglycan aggregates were prepared by reaction of the proteoglycan monomer with exogenous hyaluronic acid. 125I-labeled LDL-proteoglycan complexes were formed in the presence of 30 mM Ca2+ and incubated with macrophages. At equivalent uronic acid levels in the proteoglycans the degradation of 125I-labeled LDL contained in the native proteoglycan aggregate complex was 3.7-7.5-fold greater than the degradation of the lipoprotein in the proteoglycan monomer complex. Degradation of 125I-LDL in the in vitro aggregate complex, while higher than that in the monomer complex, was markedly less than that in the native aggregate complex. The larger size and the greater complex-forming ability of the native proteoglycan aggregate might account for the greater capacity of the aggregate to promote LDL degradation in macrophages. The proteoglycan-stimulated degradation of LDL produced a marked increase in cholesteryl ester synthesis and content in macrophages. The LDL-proteoglycan complex was degraded with saturation kinetics, suggesting that these complexes are internalized through high-affinity receptors. Degradation was inhibited by the lysosomotropic agent, chloroquine. Acetyl-LDL, but not native LDL, competitively inhibited the degradation of the 125I-LDL component of the complex. Polyanionic compounds such as polyinosinic acid and fucoidin, while completely blocking the acetyl-LDL-stimulated cholesteryl ester formation, had no effect on the proteoglycan aggregate-stimulated cholesterol esterification. This suggests that LDL-proteoglycan complex and acetyl-LDL are not entering the cells through the same receptor pathway. These results demonstrate that the interaction of LDL with arterial wall proteoglycan aggregates results in marked cholesteryl ester accumulation in macrophages, a process likely to favor foam cell formation. A role for arterial proteoglycans in atherosclerosis is obvious.
Asunto(s)
Ésteres del Colesterol/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Proteoglicanos/metabolismo , Animales , Femenino , Cinética , Ratones , Poli I/farmacología , Polisacáridos/farmacologíaRESUMEN
Proteoglycans are considered to facilitate lipid accumulation in the arterial wall, as part of the injury and repair process in atherogenesis. The present study determined (1) characteristics of arterial tissue chondroitin sulfate proteoglycan (CS-PG) monomers of versican type that vary in binding affinity to low-density lipoproteins (LDL), and (2) the ability of these variants to modulate LDL metabolism by macrophages. A large CS-PG devoid of dermatan sulfate (DS) was isolated and purified from bovine aorta intima-media under dissociative conditions. The proteoglycan was further subfractionated by LDL affinity chromatography into CS-PGI and CS-PGII variants, the former eluting at 0.1 M NaCl and the latter at 1.0 M NaCl. The core protein of both variants had a similar molecular mass (1.7 x 10(5). However, CS-PGII contained more glycosaminoglycan (GAG) chains (30 vs. 25) with higher average molecular mass (4.2 x 10(4) vs. 3.8 x 10(4)) than CS-PGI. Furthermore, CS-PGII contained a relatively higher proportion of CS6-sulfate to CS4-sulfate (65: 35 vs. 52: 48). Sulfate-to-hexosamine molar ratio of GAG measured approximately 1 in both variants. In terms of metabolism by macrophages, when compared to complex of LDL and CS-PGI, complex of LDL and CS-PGII produced consistent increase in degradation (10.3-fold vs. 8.4-fold over native LDL) and cell association (16.3-fold vs. 10.2-fold over native LDL) of the ligand, and stimulation of cholesteryl ester synthesis (8.4-fold vs. 6.4-fold over native LDL). CS-PGII was as potent as native CS/DS-PG aggregate, which is a complex made of proteoglycan monomers, hyaluronate, and link protein(s), in stimulating the above activities in macrophages. Thus, variations in LDL-binding affinity of CS-PG can potentially modulate the lipid accumulation in atherogenesis.