RESUMEN

The discovery of the ABCA1 lipid transporter has generated interest in modulating human plasma HDL levels and atherogenic risk by enhancing ABCA1 gene expression. To determine if increased ABCA1 expression modulates HDL metabolism in vivo, we generated transgenic mice that overexpress human ABCA1 (hABCA1-Tg). Hepatic and macrophage expression of hABCA1 enhanced macrophage cholesterol efflux to apoA-I; increased plasma cholesterol, cholesteryl esters (CEs), free cholesterol, phospholipids, HDL cholesterol, and apoA-I and apoB levels; and led to the accumulation of apoE-rich HDL1. ABCA1 transgene expression delayed 125I-apoA-I catabolism in both liver and kidney, leading to increased plasma apoA-I levels, but had no effect on apoB secretion after infusion of Triton WR1339. Although the plasma clearance of HDL-CE was not significantly altered in hABCA1-Tg mice, the net hepatic delivery of exogenous 3H-CEt-HDL, which is dependent on the HDL pool size, was increased 1.5-fold. In addition, the cholesterol and phospholipid concentrations in hABCA1-Tg bile were increased 1.8-fold. These studies show that steady-state overexpression of ABCA1 in vivo (a) raises plasma apoB levels without altering apoB secretion and (b) raises plasma HDL-C and apoA-I levels, facilitating hepatic reverse cholesterol transport and biliary cholesterol excretion. Similar metabolic changes may modify atherogenic risk in humans.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Bilis/metabolismo , Colesterol/metabolismo , Hiperlipoproteinemias/etiología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Apolipoproteínas/sangre , Bilis/química , Colesterol/análisis , Regulación de la Expresión Génica , Humanos , Hiperlipoproteinemias/sangre , Hiperlipoproteinemias/metabolismo , Lípidos/sangre , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Transgénicos

RESUMEN

To evaluate the biochemical and molecular mechanisms leading to glomerulosclerosis and the variable development of atherosclerosis in patients with familial lecithin cholesterol acyl transferase (LCAT) deficiency, we generated LCAT knockout (KO) mice and cross-bred them with apolipoprotein (apo) E KO, low density lipoprotein receptor (LDLr) KO, and cholesteryl ester transfer protein transgenic mice. LCAT-KO mice had normochromic normocytic anemia with increased reticulocyte and target cell counts as well as decreased red blood cell osmotic fragility. A subset of LCAT-KO mice accumulated lipoprotein X and developed proteinuria and glomerulosclerosis characterized by mesangial cell proliferation, sclerosis, lipid accumulation, and deposition of electron dense material throughout the glomeruli. LCAT deficiency reduced the plasma high density lipoprotein (HDL) cholesterol (-70 to -94%) and non-HDL cholesterol (-48 to -85%) levels in control, apoE-KO, LDLr-KO, and cholesteryl ester transfer protein-Tg mice. Transcriptome and Western blot analysis demonstrated up-regulation of hepatic LDLr and apoE expression in LCAT-KO mice. Despite decreased HDL, aortic atherosclerosis was significantly reduced (-35% to -99%) in all mouse models with LCAT deficiency. Our studies indicate (i) that the plasma levels of apoB containing lipoproteins rather than HDL may determine the atherogenic risk of patients with hypoalphalipoproteinemia due to LCAT deficiency and (ii) a potential etiological role for lipoproteins X in the development of glomerulosclerosis in LCAT deficiency. The availability of LCAT-KO mice characterized by lipid, hematologic, and renal abnormalities similar to familial LCAT deficiency patients will permit future evaluation of LCAT gene transfer as a possible treatment for glomerulosclerosis in LCAT-deficient states.

Asunto(s)

Arteriosclerosis/enzimología , Glomeruloesclerosis Focal y Segmentaria/enzimología , Fosfatidilcolina-Esterol O-Aciltransferasa/fisiología , Animales , Arteriosclerosis/fisiopatología , Secuencia de Bases , Cartilla de ADN , Glomeruloesclerosis Focal y Segmentaria/fisiopatología , Riñón/fisiopatología , Lípidos/sangre , Lipoproteínas/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , ARN Mensajero/genética

RESUMEN

Expression of human lecithin cholesterol acyltransferase (LCAT) in mice (LCAT-Tg) leads to increased high density lipoprotein (HDL) cholesterol levels but paradoxically, enhanced atherosclerosis. We have hypothesized that the absence of cholesteryl ester transfer protein (CETP) in LCAT-Tg mice facilitates the accumulation of dysfunctional HDL leading to impaired reverse cholesterol transport and the development of a pro-atherogenic state. To test this hypothesis we cross-bred LCAT-Tg with CETP-Tg mice. On both regular chow and high fat, high cholesterol diets, expression of CETP in LCAT-Tg mice reduced total cholesterol (-39% and -13%, respectively; p < 0.05), reflecting a decrease in HDL cholesterol levels. CETP normalized both the plasma clearance of [(3)H]cholesteryl esters ([(3)H]CE) from HDL (fractional catabolic rate in days(-1): LCAT-Tg = 3.7 +/- 0.34, LCATxCETP-Tg = 6.1 +/- 0.16, and controls = 6.4 +/- 0.16) as well as the liver uptake of [(3)H]CE from HDL (LCAT-Tg = 36%, LCATxCETP-Tg = 65%, and controls = 63%) in LCAT-Tg mice. On the pro-atherogenic diet the mean aortic lesion area was reduced by 41% in LCATxCETP-Tg (21.2 +/- 2.0 micrometer(2) x 10(3)) compared with LCAT-Tg mice (35.7 +/- 2.0 micrometer(2) x 10(3); p < 0.001). Adenovirus-mediated expression of scavenger receptor class B (SR-BI) failed to normalize the plasma clearance and liver uptake of [(3)H]CE from LCAT-Tg HDL. Thus, the ability of SR-BI to facilitate the selective uptake of CE from LCAT-Tg HDL is impaired, indicating a potential mechanism leading to impaired reverse cholesterol transport and atherosclerosis in these animals. We conclude that CETP expression reduces atherosclerosis in LCAT-Tg mice by restoring the functional properties of LCAT-Tg mouse HDL and promoting the hepatic uptake of HDL-CE. These findings provide definitive in vivo evidence supporting the proposed anti-atherogenic role of CETP in facilitating HDL-mediated reverse cholesterol transport and demonstrate that CETP expression is beneficial in pro-atherogenic states that result from impaired reverse cholesterol transport.

Asunto(s)

Enfermedades de la Aorta/prevención & control , Arteriosclerosis/prevención & control , Proteínas Portadoras/fisiología , Glicoproteínas , Lipoproteínas HDL/fisiología , Esterol O-Aciltransferasa/fisiología , Animales , Proteínas de Transferencia de Ésteres de Colesterol , Ésteres del Colesterol/metabolismo , Femenino , Humanos , Lipoproteínas HDL/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Esterol O-Aciltransferasa/genética

RESUMEN

Familial hypercholesterolemia (FH), a disease caused by a variety of mutations in the low density lipoprotein receptor (LDLr) gene, leads not only to elevated LDL-cholesterol (C) concentrations but to reduced high density lipoprotein (HDL)-C and apolipoprotein (apo) A-I concentrations as well. The reductions in HDL-C and apoA-I are the consequence of the combined metabolic defects of increased apoA-I catabolism and decreased apoA-I synthesis. The present studies were designed to test the hypothesis that overexpression of human lecithin:cholesterol acyltransferase (hLCAT), a pivotal enzyme involved in HDL metabolism, in LDLr defective rabbits would increase HDL-C and apoA-I concentrations. Two groups of hLCAT transgenic rabbits were established: 1) hLCAT+/LDLr heterozygotes (LDLr+/-) and 2) hLCAT+/LDLr homozygotes (LDLr-/-). Data for hLCAT+ rabbits were compared to those of nontransgenic (hLCAT-) rabbits of the same LDLr status. In LDLr+/- rabbits, HDL-C and apoA-I concentrations (mg/dl), respectively, were significantly greater in hLCAT+ (62 +/- 8, 59 +/- 4) relative to hLCAT- rabbits (21 +/- 1, 26 +/- 2). This was, likewise, the case when hLCAT+/ LDLr-/- (27 +/- 2, 19 +/- 6) and hLCAT-/LDLr-/- (5 +/- 1, 6 +/- 2) rabbits were compared. Kinetic experiments demonstrated that the fractional catabolic rate (FCR, d(-1)) of apoA-I was substantially delayed in hLCAT+ (0.376 +/- 0.025) versus hLCAT- (0.588) LDLr+/- rabbits, as well as in hLCAT+ (0.666 +/- 0.033) versus hLCAT- (1.194 +/- 0.138) LDLr-/- rabbits. ApoA-I production rate (PR, mg x kg x d(-1)) was greater in both hLCAT+/LDLr+/- (10 +/- 2 vs. 6) and hLCAT+/LDLr-/- (9 +/- 1 vs. 4 +/- 1) rabbits. Significant correlations (P < 0.02) were observed between plasma LCAT activity and HDL-C (r = 0.857), apoA-I FCR (r = -0.774), and apoA-I PR (r = 0.771), while HDL-C correlated with both apoA-I FCR (-0.812) and PR (0.751). In summary, these data indicate that hLCAT overexpression in LDLr defective rabbits increases HDL-C and apoA-I concentrations by both decreasing apoA-I catabolism and increasing apoA-I synthesis, thus correcting the metabolic defects responsible for the hypoalphalipoproteinemia observed in LDLr deficiency.

Asunto(s)

Hipolipoproteinemias/terapia , Lipoproteínas HDL/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Receptores de LDL/deficiencia , Animales , Animales Modificados Genéticamente , Apolipoproteína A-I/sangre , Secuencia de Bases , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Expresión Génica , Terapia Genética , Heterocigoto , Homocigoto , Humanos , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/terapia , Hipolipoproteinemias/sangre , Hipolipoproteinemias/genética , Cinética , Lípidos/sangre , Lipoproteínas/sangre , Mutación , Conejos , Receptores de LDL/genética

RESUMEN

In order to evaluate the coordinate role that hepatic lipase (HL) and lecithin:cholesterol acyltransferase (LCAT) play in modulating HDL particle heterogeneity and function in vivo we utilized recombinant adenovirus to express HL in control and LCAT transgenic mice. Adenovirus-mediated expression of human HL in control (n = 4, LCAT activity = 42 +/- 1 nmol/ml per h) and LCAT-tg mice (n = 4, LCAT activity = 3566 +/- 93 nmol/ml per h) resulted in post heparin HL activities of 24,358 +/- 6080 and 27,266 +/- 7985 nmol/ml per min, respectively. Overexpression of HL led to significant reductions in total cholesterol, phospholipids, and HDL cholesterol in both LCAT-tg (62, 62, and 63%, P < 0.05) and control mice (68, 63, and 78%, P < 0.01) as well as to the formation of more homogenous HDL. However, compared to control animals, the reductions in the plasma concentrations of HDL-cholesterol and apoA-I were less in LCAT-tg mice (HDL-cholesterol: -62 +/- 15% vs. -78 +/- 15%, P = 0.18; apoA-I: -36 +/- 7% vs. -76 +/- 8%, P < 0.0005). Gel filtration analysis revealed that in LCAT-tg mice the apoE-rich HDL1 was preferentially reduced by expression of HL in vivo. Compared to control mice the reduction in the apoA-I/A-II HDL in transgenic mice was significantly less indicating that a subset of HDL in LCAT transgenic mice are resistant to the action of HL. These combined data support a role for both HL and LCAT in modulating HDL heterogeneity and function, properties which may ultimately affect the ability of LCAT transgenic mouse HDL to function in the process of reverse cholesterol transport.

Asunto(s)

Adenoviridae/genética , Lipasa/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Animales , Apolipoproteína A-I/sangre , Secuencia de Bases , Colesterol/sangre , Cartilla de ADN/genética , Expresión Génica , Humanos , Lipasa/sangre , Lípidos/sangre , Lipoproteínas HDL/sangre , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre

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/metabolismo

RESUMEN

We have established a mouse model for human LCAT deficiency by performing targeted disruption of the LCAT gene in mouse embryonic stem cells. Homozygous LCAT-deficient mice were healthy at birth and fertile. Compared with age-matched wild-type littermates, the LCAT activity in heterozygous and homozygous knockout mice was reduced by 30 and 99%, respectively. LCAT deficiency resulted in significant reductions in the plasma concentrations of total cholesterol, HDL cholesterol, and apoA-I in both LCAT -/- mice (25, 7, and 12%; p < 0. 001 of normal) and LCAT +/- mice (65 and 59%; p < 0.001 and 81%; not significant, p = 0.17 of normal). In addition, plasma triglycerides were significantly higher (212% of normal; p < 0.01) in male homozygous knockout mice compared with wild-type animals but remained normal in female knockout LCAT mice. Analyses of plasma lipoproteins by fast protein liquid chromatography and two-dimensional gel electrophoresis demonstrated the presence of heterogenous prebeta-migrating HDL, as well as triglyceride-enriched very low density lipoprotein. After 3 weeks on a high-fat high-cholesterol diet, LCAT -/- mice had significantly lower plasma concentrations of total cholesterol, reflecting reduced levels of both proatherogenic apoB-containing lipoproteins as well as HDL, compared with controls. Thus, we demonstrate for the first time that the absence of LCAT attenuates the rise of apoB-containing lipoproteins in response to dietary cholesterol. No evidence of corneal opacities or renal insufficiency was detected in 4-month-old homozygous knockout mice. The availability of a homozygous animal model for human LCAT deficiency states will permit further evaluation of the role that LCAT plays in atherosclerosis as well as the feasibility of performing gene transfer in human LCAT deficiency states.

Asunto(s)

Modelos Animales de Enfermedad , Deficiencia de la Lecitina Colesterol Aciltransferasa/genética , Animales , Femenino , Eliminación de Gen , Marcación de Gen , Humanos , Masculino , Ratones , Ratones Noqueados

RESUMEN

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 , Conejos

RESUMEN

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/patología

RESUMEN

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 , Conejos

RESUMEN

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.

Asunto(s)

Apolipoproteínas/sangre , HDL-Colesterol/sangre , Expresión Génica , Hiperlipoproteinemias/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Animales , Animales Modificados Genéticamente , Northern Blotting , Encéfalo/enzimología , Colesterol/sangre , Ésteres del Colesterol/sangre , Embrión de Mamíferos , Femenino , Humanos , Hiperlipoproteinemias/sangre , Hiperlipoproteinemias/enzimología , Hígado/enzimología , Masculino , Ratones , Especificidad de Órganos , Fosfatidilcolina-Esterol O-Aciltransferasa/biosíntesis , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfolípidos/sangre , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Conejos , Valores de Referencia , Triglicéridos/sangre

RESUMEN

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 Nucleicos

RESUMEN

Two transgenic rabbits which carried human apolipoprotein A-1 (apo A-1) cDNA under mouse ribosomal protein L/32 promoter were obtained. The effectiveness of transgenosis was confirmed by DNA dot/blot and Southern blot hybridizations. Both transgenic animals had paralyses of fore or fore and high limbs. Electron microscopy demonstrated distinct degradative changes of those parts of spinal cord which were responsible for leg skeletal muscle innervation. RNA dot/blot hybridization showed transgene expression in liver and brain but not in kidney of adult transgenic animal. However, analysis of blood serum lipids and immunochemical determinations gave no indications of the presence of human apo A-1 in adult transgenic rabbit. The data obtained allow us to suggest that the observed pathology was due to interference of native and foreign protein products of apo A-1 gene expression in CNS in the course of embryo development. This suggestion was supported by results of in situ hybridization of 5- and 9-week human embryo sections with apo A-1 cDNA, showing effective expression of apo A-1 gene in neural cells of CNS. Results of transgenosis may be viewed as modeling of the neurological syndrome of human Tangier disease.

Asunto(s)

Apolipoproteína A-I/genética , ADN , Enfermedades del Sistema Nervioso/genética , Enfermedad de Tangier/genética , Animales , Animales Modificados Genéticamente , Apolipoproteína A-I/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Humanos , Microscopía Electrónica , Modelos Neurológicos , Músculos/inervación , Músculos/metabolismo , Músculos/ultraestructura , Enfermedades del Sistema Nervioso/complicaciones , Hibridación de Ácido Nucleico , Regiones Promotoras Genéticas , Conejos , Enfermedad de Tangier/complicaciones , Distribución Tisular

Asunto(s)

Carcinoma/cirugía , Hemorragia Gastrointestinal/cirugía , Neoplasias Gástricas/cirugía , Rotura Gástrica/cirugía , Carcinoma/complicaciones , Hemorragia Gastrointestinal/etiología , Humanos , Masculino , Persona de Mediana Edad , Rotura Espontánea , Neoplasias Gástricas/complicaciones , Rotura Gástrica/etiología

RESUMEN

It was found that in patients with gastroduodenal hemorrhage the number of serotonin-, histamine-, and melatonin-producing apudocytes in the gastric mucosa increases while the number of cells producing gastrin, adrenaline, and noradrenaline reduces. In timely arrest of hemorrhage, the primarily increased number of melatonin-producing apudocytes diminishes with time from the onset of bleeding and reaches normal values gradually. The essential differences in the content of melatonin- and noradrenaline- producing apudocytes in patients with and without hemorrhage allow this morphological sign to be used as diagnostic and prognostic criteria in gastroduodenal hemorrhages.

Asunto(s)

Células APUD/patología , Úlcera Duodenal/complicaciones , Úlcera Péptica Hemorrágica/patología , Antro Pilórico/patología , Úlcera Gástrica/complicaciones , Células APUD/metabolismo , Adulto , Anciano , Catecolaminas/biosíntesis , Recuento de Células , Úlcera Duodenal/patología , Femenino , Gastrinas/metabolismo , Hormonas Ectópicas/metabolismo , Humanos , Masculino , Melatonina/metabolismo , Persona de Mediana Edad , Úlcera Gástrica/patología

Asunto(s)

Cruzamiento , ADN Recombinante , Ingeniería Genética/métodos , Ratones Transgénicos/genética , Óvulo/trasplante , Animales , Clonación Molecular , Regulación de la Expresión Génica , Ratones

Asunto(s)

Ingeniería Genética , Oncogenes , Animales , ADN/genética , Enzimas de Restricción del ADN , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Hibridación de Ácido Nucleico , Plásmidos , Transformación Genética

RESUMEN

The human growth hormone gene, containing mouse metallothionein gene promotor, was injected into the male pronuclei of fertilized mouse ova. The progeny of transgenic mice included animals with both accelerated and inhibited growth. Radioimmunochemical analysis has revealed human growth hormone synthesis in both groups of transgenic mice. The molecular weight of the hormone synthesized in liver cells was 25,000 daltons. A possible mechanisms of foreign hormone effect on the growth of transgenic mice is discussed.

Asunto(s)

Regulación de la Expresión Génica , Ingeniería Genética , Hormona del Crecimiento/genética , Crecimiento , Animales , ADN/genética , Femenino , Humanos , Masculino , Metalotioneína/genética , Ratones , Microinyecciones , Regiones Promotoras Genéticas , Transformación Genética

Asunto(s)

Ingeniería Genética , Simplexvirus/genética , Timidina Quinasa/genética , Transformación Genética , Cigoto , Animales , Ratones , Microinyecciones , Simplexvirus/enzimología

RESUMEN

In mice obtained after microinjection into the male pronucleus of fertilized eggs of the plasmid, containing the bacterial gene of dihydrofolate reductase (DHFR), under the control of the early promotor of the simian virus 40 (SV40), an integration of the foreign DNA into the mouse genome is found. About 30% of the treated animals contain the integrated plasmid DNA sequences, i.e. are transgenic. In 2 of 7 mice, containing the introduced plasmid in their genome, the methotrexate-resistant DHFR activity is found in the kidney and spleen, which may be due to the expression of gene DHFR. The plasmid DNA sequences and the ability to synthesise the methotrexate-resistant enzyme DHFR are transmitted to the next generation of mice.

Asunto(s)

Genes Bacterianos , Tetrahidrofolato Deshidrogenasa/genética , Transformación Genética , Animales , ADN Bacteriano/genética , Femenino , Antagonistas del Ácido Fólico , Regulación de la Expresión Génica , Masculino , Metotrexato/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Hibridación de Ácido Nucleico , Plásmidos , Regiones Promotoras Genéticas , Virus 40 de los Simios/genética