RESUMEN
A subset of patients with high plasma HDL concentrations have enhanced rather than reduced atherosclerosis. We have developed a new transgenic mouse model overexpressing human lecithin-cholesteryl acyltransferase (LCAT) that has elevated HDL and increased diet-induced atherosclerosis. LCAT transgenic mouse HDLs are abnormal in both composition and function. Liver uptake of [3H]cholesteryl ether incorporated in transgenic mouse HDL was reduced by 41% compared with control HDL, indicating ineffective transport of HDL-cholesterol to the liver and impaired reverse cholesterol transport. Analysis of this LCAT-transgenic mouse model provides in vivo evidence for dysfunctional HDL as a potential mechanism leading to increased atherosclerosis in the presence of high plasma HDL levels.
Asunto(s)
Arteriosclerosis/sangre , Lipoproteínas HDL/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/biosíntesis , Animales , Aorta/patología , Arteriosclerosis/enzimología , Arteriosclerosis/patología , Colesterol/sangre , Dieta Aterogénica , Modelos Animales de Enfermedad , Femenino , Humanos , Lípidos/sangre , Lipoproteínas HDL/química , Lipoproteínas HDL/fisiología , Masculino , Ratones , Ratones Transgénicos , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismoRESUMEN
Monoclonal free light chains are found in the serum and urine of patients with B-cell proliferative disorders, including multiple myeloma. Measuring free light chains in serum is useful for the diagnosis and monitoring of free light chains diseases. Moreover it could be interesting in the monitoring of treated multiple myeloma with complete immunoglobulin and of monoclonal gammapathy of undetermined significance (MGUS). The goal of our work was to analyze a large cohort of patients with multiple myeloma or MGUS from January 2003 to August 2006 in order to better understand the interest of free light chains.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina/sangre , Mieloma Múltiple/sangre , Paraproteinemias/sangre , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/inmunología , Paraproteinemias/inmunología , Estudios RetrospectivosRESUMEN
Lecithin cholesterol acyltransferase (LCAT) is an enzyme involved in the intravascular metabolism of high density lipoproteins (HDLs). Overexpression of human LCAT (hLCAT) in transgenic rabbits leads to gene dose-dependent increases of total and HDL cholesterol concentrations. To elucidate the mechanisms responsible for this effect, 131I-HDL apoA-I kinetics were assessed in age- and sex-matched groups of rabbits (n=3 each) with high, low, or no hLCAT expression. Mean total and HDL cholesterol concentrations (mg/dl), respectively, were 162+/-18 and 121+/-12 for high expressors (HE), 55+/-6 and 55+/-10 for low expressors (LE), and 29+/-2 and 28+/-4 for controls. Fast protein liquid chromatography analysis of plasma revealed that the HDL of both HE and LE were cholesteryl ester and phospholipid enriched, as compared with controls, with the greatest differences noted between HE and controls. These compositional changes resulted in an incremental shift in apparent HDL particle size which correlated directly with the level of hLCAT expression, such that HE had the largest HDL particles and controls the smallest. In vivo kinetic experiments demonstrated that the fractional catabolic rate(FCR, d(-1)) of apoA-I was slowest in HE (0.328+/-0.03) followed by LE (0.408+/-0.01) and, lastly, by controls (0.528+/-0.04). ApoA-I FCR was inversely associated with HDL cholesterol level (r=-0.851,P<0.01) and hLCAT activity (r=-0.816, P<0.01). These data indicate that fractional catabolic rate is the predominant mechanism by which hLCAT overexpression differentially modulates HDL concentrations in this animal model. We hypothesize that LCAT-induced changes in HDL composition and size ultimately reduce apoA-I catabolism by altering apoA-I conformation and/or HDL particle regeneration.
Asunto(s)
Apolipoproteína A-I/metabolismo , HDL-Colesterol/sangre , Hiperlipoproteinemias/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/biosíntesis , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Ésteres del Colesterol/sangre , Cromatografía Líquida de Alta Presión , Expresión Génica , Humanos , Hiperlipoproteinemias/sangre , Hiperlipoproteinemias/metabolismo , Cinética , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfolípidos/sangre , ConejosRESUMEN
BACKGROUND AND METHODS: Hepatic lipase (HL) is an enzyme which hydrolyzes triglycerides from plasma lipoproteins and thus takes part in the metabolism of triglyceride-rich lipoprotein remnants and high density lipoproteins. The search described here concentrated on the description of the double invalidation of the HL and LDL receptor genes in mice in order to better understand the possible role of HL in combined hyperlipidemia/hyperalphalipoproteinemia and development of atherosclerosis. RESULTS: We show here that mice lacking both endogenous HL and LDL receptor (HL-/-:LDLR-/-) dramatically increased their plasma triglyceride-rich lipoproteins and their remnants as a consequence of reduced liver uptake. This result is strenghthened by the fact that HL-/-:LDLR-/- were found to overexpress LRP, LSR, and apoE genes. Interestingly, HL-/-:LDLR-/- mice showed premature spontaneous atherosclerosis and aortic lesions from 1-year-old animals were two-fold larger than those of LDLR-/- single mutants. We confirmed that HL-/- and wild-type mice did not develop atherosclerosis lesion even 1 year after birth. CONCLUSIONS: Analysis of this double HL-LDLR knockout mouse model provides in vivo evidence that HL has a major role in the clearance of TRL remnants when LDLR is deficient and in the reduction of the development of atherosclerosis.
Asunto(s)
Aterosclerosis/genética , Hiperlipidemias/genética , Hiperlipoproteinemia Tipo I/genética , Lipasa/deficiencia , Receptores de LDL/deficiencia , Animales , Aorta/patología , Aterosclerosis/patología , Hiperlipidemias/patología , Hiperlipoproteinemia Tipo I/patología , Lipasa/genética , Lípidos/sangre , Lipoproteínas/sangre , Hígado/metabolismo , Ratones , Ratones Noqueados , Receptores de LDL/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The objectives of this study were to evaluate the outcome after polychemotherapy for patients with primary cutaneous large-cell lymphomas (PCLL) and to validate the recently proposed immunohistologic classification of cutaneous lymphomas. Among 140 patients with positive skin biopsies included in the LNH87 protocol (for treatment of aggressive lymphomas), 49 patients met the criteria of PCLL. Characteristics were: sex ratio M/F, 2.3; age 18 to 83 years (median, 52), peripheral lymph nodes, n=22; diffuse disease, n=12; median tumor size, 4.5 cm; elevated lactate dehydrogenase, n=9; ECOG: 0/1, n=49. Histology was: follicular center B cell, n=23; B-lymphoblastic, n=1; anaplastic large-cell lymphoma, n=14 (T cell phenotype n=8); CD30- T cell lymphoma, n=11. All patients received polychemotherapy: under 70 years, ACVBP (three to four cycles and consolidation for 6 months) n=25; mBACOD (eight cycles) n=16; over 70 years, C(T)VP (six cycles) n=8. Radiation therapy was not included in the protocol. With a median follow-up of 5 years, 24/49 patients had relapsed, with 20 skin relapses. Event-free (EFS) and overall survival (OS) at 5 years were, respectively, 50 and 77%. Significant adverse prognostic factors were: histology (CD30- T cell lymphoma) and diffuse cutaneous disease (>10% of skin). The presence of nodal involvement was only significant for EFS. When compared to 140 non-cutaneous lymphoma patients included in the same trial and fully matched for the main clinical characteristics, OS was similar. In conclusion, PCLL behaves like other localized B or T cell extranodal lymphomas with the same prognostic factors (LDH, ECOG, age) except for CD30+ PCLL which have a very good prognosis.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma no Hodgkin/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Bleomicina/administración & dosificación , Estudios de Casos y Controles , Ciclofosfamida/administración & dosificación , Dexametasona/administración & dosificación , Doxorrubicina/administración & dosificación , Femenino , Humanos , L-Lactato Deshidrogenasa/metabolismo , Leucovorina/administración & dosificación , Masculino , Metotrexato/administración & dosificación , Persona de Mediana Edad , Prednisona/administración & dosificación , Pronóstico , Estudios Prospectivos , Resultado del Tratamiento , Vincristina/administración & dosificación , Vindesina/administración & dosificaciónRESUMEN
Lecithin-cholesterol acyltransferase (LCAT) is the major enzyme involved in the esterification of cholesterol in circulating plasma lipoproteins. In the present study, we describe the molecular defects in the LCAT gene and in lipoprotein metabolism of a 34-year-old patient presenting with features of classic familial LCAT deficiency. DNA sequencing revealed two separate point mutations in exon 3 of the patient's LCAT gene: a C to A substitution converting Tyr(83) to a Stop and a C to T transition converting an Arg(99) to a Cys. Digestion of patient PCR-amplified DNA with the restriction enzymes AccI and AciI established that the patient was a compound heterozygote for both mutations. In vitro expression of LCAT (Arg(99)-->Cys) in human embryonic kidney-293 cells demonstrated reduced expression, as well as reduced secretion and/or increased intracellular degradation of the mutant enzyme with significantly decreased alpha-LCAT specific activity, thus, establishing the functional significance of the LCAT (Arg(99)-->Cys) mutation. The plasma cholesterol esterification rate (CER, 2+/-0.3 nmol/ml/h), alpha-LCAT activity (2.9+/-0.1 nmol/ml/h) and LCAT concentration (0.3+/-0.1 microg/ml) were 2.9%, 2.3% and 6.1% that of normal subjects, respectively. Analysis of the patient's plasma lipid profile revealed reduced plasma concentrations of total cholesterol (111+/-0.5 mg/dl), HDL cholesterol (1.6+/-0.2 mg/dl), apolipoprotein (apo) A-I (52+/-4 mg/dl) and apo A-II (11+/-0.5 mg/dl). Nevertheless, for the first time, we demonstrate that the LCAT-deficient plasma is as efficient as control plasma in cholesterol efflux experiments performed with [(3)H]-cholesterol loaded fibroblasts. This result could explain the absence of premature atherosclerosis in this LCAT-deficient patient.
Asunto(s)
Colesterol/sangre , Deficiencia de la Lecitina Colesterol Aciltransferasa/sangre , Adulto , Apolipoproteínas/sangre , Arginina/metabolismo , Células Cultivadas , Colesterol/genética , Cistina/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/genética , Fibroblastos , Humanos , Cinética , Deficiencia de la Lecitina Colesterol Aciltransferasa/genética , Lípidos/sangre , Lipoproteínas/sangre , Masculino , Fenotipo , Mutación Puntual , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: Increased HDL-cholesterol (HDL-C) concentrations have been associated with lower coronary heart disease risk. On the other hand, dietary fats are known to influence the fatty acid profile of plasma lipids, including phospholipids that are substrates of lecithin cholesterol acyltransferase (LCAT), an important enzyme in HDL metabolism. The purpose of this study was to examine the association between the saturated fatty acid (SFA) intake and LCAT activity. DESIGN: An interventional study was performed in a monk community of 25 men. SETTING: A French monk community, South West of France. SUBJECTS AND INTERVENTIONS: The basal diet of the study cohort contained SFA in a proportion of 13.5% of their total energy intake (TEI). They were submitted to two experimental isocaloric diets containing either 8.4% of the TEI in SFA (diet A) or 11% (diet B), each lasting 5 weeks. RESULTS: The elevation of SFA in diet B was mainly obtained by decreasing carbohydrates. The only significant difference among total fats between diets A and B was the myristic acid content (0.6 and 1.2% of TEI, respectively). The elevation in SFA in diet B resulted in a significant increase of HDL-C (P<0.04), while plasma apo A-I concentration and LCAT activity both decreased (P<0.02). CONCLUSION: Altogether, these results are consistent with a negative effect of SFA on reverse cholesterol transport.
Asunto(s)
Apolipoproteína A-I/sangre , HDL-Colesterol/sangre , Grasas de la Dieta/administración & dosificación , Ácidos Grasos/administración & dosificación , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Adulto , Anciano , Estudios de Cohortes , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Persona de Mediana Edad , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolinas/sangreRESUMEN
Lecithin:cholesterol acyltransferase (LCAT) is an enzyme well known for its involvement in the intravascular metabolism of high density lipoproteins; however, its role in the regulation of apolipoprotein (apo) B-containing lipoproteins remains elusive. The present study was designed to investigate the metabolic mechanisms responsible for the differential lipoprotein response observed between cholesterol-fed hLCAT transgenic and control rabbits. 131I-labeled HDL apoA-I and 125I-labeled LDL kinetics were assessed in age- and sex-matched groups of rabbits with high (HE), low (LE), or no hLCAT expression after 6 weeks on a 0.3% cholesterol diet. In HE, the mean total cholesterol concentration on this diet, mg/dl (230 +/- 50), was not significantly different from that of either LE (313 +/- 46) or controls (332 +/- 52) due to the elevated level of HDL-C observed in HE (127 +/- 19), as compared with both LE (100 +/- 33) and controls (31 +/- 4). In contrast, the mean nonHDL-C concentration for HE (103 +/- 33) was much lower than that for either LE (213 +/- 39) or controls (301 +/- 55). FPLC analysis of plasma confirmed that HDL was the predominant lipoprotein class in HE on the cholesterol diet, whereas cholesteryl ester-rich, apoB-containing lipoproteins characterized the plasma of LE and, most notably, of controls. In vivo kinetic experiments demonstrated that the differences in HDL levels noted between the three groups were attributable to distinctive rates of apoA-I catabolism, with the mean fractional catabolic rate (FCR, d-1) of apoA-I slowest in HE (0.282 +/- 0.03), followed by LE (0.340 +/- 0.01) and controls (0.496 +/- 0.04). A similar, but opposite, pattern was observed for nonHDL-C levels and LDL metabolism (h-1), such that HE had the lowest nonHDL-C levels with the fastest rate of clearance (0.131 +/- 0.027), followed by LE (0.057 +/- 0.009) and controls (0.031 +/- 0.001). Strong correlations were noted between LCAT activity and both apoA-I (r= -0.868, P < 0.01) and LDL (r = 0.670, P = 0.06) FCR, indicating that LCAT activity played a major role in the mediation of lipoprotein metabolism. In summary, these data are the first to show that LCAT overexpression can regulate both LDL and HDL metabolism in cholesterol-fed rabbits and provide a potential explanation for the prevention of diet-induced atherosclerosis observed in our previous study.
Asunto(s)
Colesterol/administración & dosificación , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Animales , Animales Modificados Genéticamente , Apolipoproteína A-I/farmacocinética , Apolipoproteínas B/farmacocinética , Colesterol/sangre , Ésteres del Colesterol/sangre , Cromatografía en Gel , Dosificación de Gen , Humanos , Radioisótopos de Yodo/metabolismo , Cinética , Hígado/enzimología , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfolípidos/sangre , ConejosRESUMEN
Lecithin cholesterol acyltransferase (LCAT) is a key enzyme which catalyzes the esterification of free cholesterol present in plasma lipoproteins. In order to evaluate the role of LCAT in HDL metabolism, a 6.2-kilobase (kb) fragment consisting of 0.851 and 1.134 kb of the 5'- and 3'-flanking regions, as well as the entire human LCAT gene, was utilized to develop transgenic mice. Three different transgenic mouse lines overexpressing human LCAT at plasma levels 11-, 14-, and 109-fold higher than non-transgenic mice were established. Northern blot hybridization analysis demonstrated that the injected 6.2-kb fragment contained the necessary DNA sequences to direct tissue specific expression of the human LCAT gene in mouse liver. Compared to age- and sex-matched controls, total cholesterol and HDL cholesterol levels were increased in all 3 transgenic mice lines by 124-218 and 123-194%, respectively, while plasma triglyceride concentrations remained similar to that of control animals. Fast protein liquid chromatography analysis of transgenic mouse plasma revealed marked increases in high density liposportin (HDL)-cholesteryl ester and phospholipid as well as the formation of larger size HDL. Thus, the majority of the increase in transgenic plasma cholesterol concentrations was due to accumulation of cholesteryl ester in HDL consistent with enhanced esterification of free cholesterol in mouse HDL by human LCAT. Plasma concentrations of apoA-I, apoA-II, and apoE were increased in high expressor homozygote mice who also demonstrated an accumulation of an apoE-rich HDL1. Like the mouse enzyme, human LCAT was found to be primarily associated with mouse HDL. Our studies demonstrate a high correlation between plasma LCAT activity and total as well as HDL cholesterol levels establishing that in mice LCAT modulates plasma HDL concentrations. Overexpression of LCAT in mice leads to HDL elevation as well as increased heterogeneity of the HDL lipoprotein particles, indicating that high levels of plasma LCAT activity may be associated with hyperalphalipoproteinemia and enhanced reverse cholesterol transport.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Hiperlipoproteinemias/genética , Lipoproteínas HDL/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/biosíntesis , Animales , HDL-Colesterol/sangre , ADN Complementario/genética , Femenino , Heterocigoto , Humanos , Lípidos/sangre , Lipoproteínas/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Lecithin:cholesterol acyltransferase (LCAT) is a key plasma enzyme in cholesterol and high density lipoprotein (HDL) metabolism. Transgenic rabbits overexpressing human LCAT had 15-fold greater plasma LCAT activity that nontransgenic control rabbits. This degree of overexpression was associated with a 6.7-fold increase in the plasma HDL cholesterol concentration in LCAT transgenic rabbits. On a 0.3% cholesterol diet, the HDL cholesterol concentrations increased from 24 +/- 1 to 39 +/- 3 mg/dl in nontransgenic control rabbits (n = 10; P < 0.05) and increased from 161 +/- 5 to 200 +/- 21 mg/dl (P < 0.001) in the LCAT transgenic rabbits (n = 9). Although the baseline non-HDL concentrations of control (4 +/- 3 mg/dl) and transgenic rabbits (18 +/- 4 mg/dl) were similar, the cholesterol-rich diet raised the non-HDL cholesterol concentrations, reflecting the atherogenic very low density, intermediate density, and low density lipoprotein particles observed by gel filtration chromatography. The non-HDL cholesterol rose to 509 +/- 57 mg/dl in controls compared with only 196 +/- 14 mg/dl in the LCAT transgenic rabbits (P < 0.005). The differences in the plasma lipoprotein response to a cholesterol-rich diet observed in the transgenic rabbits paralleled the susceptibility to developing aortic atherosclerosis. Compared with nontransgenic controls, LCAT transgenic rabbits were protected from diet-induced atherosclerosis with significant reductions determined by both quantitative planimetry (-86%; P < 0.003) and quantitative immunohistochemistry (-93%; P < 0.009). Our results establish the importance of LCAT in the metabolism of both HDL and apolipoprotein B-containing lipoprotein particles with cholesterol feeding and the response to diet-induced atherosclerosis. In addition, these findings identify LCAT as a new target for therapy to prevent atherosclerosis.
Asunto(s)
Aorta Torácica/patología , Arteriosclerosis/prevención & control , Dieta Aterogénica , Lipoproteínas/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/biosíntesis , Animales , Animales Modificados Genéticamente , Arteriosclerosis/sangre , Arteriosclerosis/patología , Colesterol/sangre , HDL-Colesterol/sangre , Terapia Genética , Humanos , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Conejos , Valores de Referencia , Análisis de Regresión , Triglicéridos/sangre , Túnica Íntima/patologíaRESUMEN
Cholesterol esterification within plasma lipoprotein particles is catalyzed by lecithin:cholesterol acyltransferase (LCAT). The impact of the overexpression of this enzyme on plasma concentrations of the different plasma lipoproteins in an animal model expressing cholesteryl ester transfer protein was evaluated by generating rabbits expressing human LCAT. A 6.2-kilobase human genomic DNA construct was injected into the pronuclei of rabbit embryos. Of the 1002 embryos that were injected, 3 founder rabbits were characterized that expressed the human LCAT gene. As in mice and humans, the principal sites of mRNA expression in these rabbits is in the liver and brain, indicating that the regulatory elements required for tissue-specific expression among these species are similar. The alpha-LCAT activity correlated with the number of copies of LCAT that integrated into the rabbit DNA. Compared with controls, the high expressor LCAT-transgenic rabbits total and high density lipoprotein (HDL) cholesterol concentrations were increased 1.5-2.5-fold with a 3.1-fold increase in the plasma cholesterol esterification rate. Analysis of the plasma lipoproteins by fast protein liquid chromatography indicates that these changes reflected an increased concentration of apolipoprotein E-enriched, HDL1-sized particles, whereas atherogenic apolipoprotein B particles disappeared from the plasma. The concentrations of plasma HDL cholesterol were highly correlated with both human LCAT mass (r = 0.93; p = 0.001) and the log LCAT activity (r = 0.94; p < 0.001) in the transgenic rabbits. These results indicate that overexpression of LCAT in the presence of cholesteryl ester transfer protein leads to both hyperalpha-lipoproteinemia and reduced concentrations of atherogenic lipoproteins.