Glucocorticoid receptor expression is down-regulated by Lp(a) lipoprotein in vascular smooth muscle cells.

Glucocorticoids have been reported to protect against atherosclerosis and have been used clinically as protective therapy for restenosis after balloon angioplasty. Recently, Lp(a) lipoprotein [Lp(a)] levels have been suggested to be an independent risk factor for atherosclerosis, although its mechanisms of action are still uncertain. To clarify this atherogenic mechanism of Lp(a), we investigated the effects of Lp(a) on glucocorticoid receptor (GR) expression in human vascular smooth muscle cells (SMC). Levels of nuclear GR in SMC began to decrease after 12-h incubation with Lp(a), to 55 +/- 8% of the control value at 48 h; binding affinity did not change. Lp(a) had no effect on estrogen receptor binding in SMC. Moreover, low, very low, and high density lipoproteins had no effect on GR binding in SMC. The effects of Lp(a) on nuclear GR in rat SMC were very similar to those in human SMC; in contrast, Lp(a) did not alter GR or estrogen receptor levels in rat endothelial cells. GR messenger RNA levels in SMC decreased after 1-h treatment with Lp(a) to 23% of the control value after 12 h. Further, the antiproliferative effect of glucocorticoids on SMC was blunted by exposure to Lp(a). We conclude that Lp(a) down-regulates GR gene expression, resulting in a decreased number of GR in SMC. These findings suggest the possibility of a novel atherogenic mechanism of Lp(a) via inhibition of a protective action of glucocorticoids on SMC.

Regulation of cholesterol uptake in the rat intestinal cell line.

A new model to study cholesterol absorption in the rat intestinal cells is described. Rat intestine epithelial cells IRD98 were incubated with mixed micelles containing bile acid, phospholipid, cholesterol or its nonabsorbable analogue, sitosterol, and trace amounts of [3H]cholesterol or [14C]sitosterol. Cholesterol and sitosterol uptake was then determined following lipid extraction; specific cholesterol uptake was determined as the difference between cholesterol and sitosterol uptake. Cholesterol, but not sitosterol, uptake was time- and dose-dependent and saturable. Loading of cells with non-lipoprotein cholesterol reduced cholesterol, but not sitosterol, uptake in a dose-dependent manner. In contrast, treatment of cells with an inhibitor of cholesterol synthesis, lovastatin, stimulated cholesterol, but not sitosterol, uptake in a dose-dependent manner. Treatment of cells with palmitic, caproic and oleic acids up-regulated specific cholesterol uptake, while linoleic and stearic acids had an opposite effect. None of the fatty acids affected sitosterol uptake.

Time response of cholesterol synthesis inhibition by compactin-related compounds. In vitro quantitation of the "escape phenomenon".

The time course of the inhibition of cholesterol synthesis by low and high doses of mevinolin and monacolin X were studied in normal human skin fibroblasts, fibroblasts without low density lipoprotein receptor and HepG2 hepatoma cells. Low doses of the inhibitors (0.2 ng/mL) caused a sharp decrease in the rate of cholesterol synthesis during the first 2-3 h, which gradually increased to about 40% during the next 6 h. Further incubation led to a decrease or stabilization of the cholesterol synthesis rate. High doses of the drugs (100 mg/mL) strongly inhibited cholesterol synthesis during the first 2-3 h, followed by a moderate increase during the next 20 h. No drug or tissue selectivity was observed.

[Regulation of cholesterol absorption in human and rat small intestine epithelial cells]. / Reguliatsiia vsasyvaniia kholesterina v épitelial'nykh kletkakh tonkogo kishechnika cheloveka i krysy.

Cholesterol absorption in human small intestine organ culture and rat small intestine epithelial cell culture IRD-98 has been studied using [14C] cholesterol, [3H] cholesterol and [14C] sitosterol. It has been found that cholesterol absorption is a dose- and time-dependent process, while sitosterol absorption is not and makes up to about 25% of the total cholesterol absorption. Cholesterol absorption appeared to be a specific process. The endocytosis inhibitor monensin decreased specific cholesterol absorption by 37%. Cholesterol absorption was examined under different conditions influencing cholesterol metabolism in the cell. Loading of IRD-98 cells with non-lipoprotein cholesterol caused a dose-dependent decrease of cholesterol absorption. The inhibitor of acyl coenzyme A:cholesterol acyl transferase (ACAT), compound Sandoz 58-035, had a similar effect on cholesterol absorption. Lovastatin, an inhibitor of 3-hydroxymethyl-3-glutaryl coenzyme A (HMG-CoA) reductase, stimulated cholesterol absorption in a dose-dependent manner. Loading of cells with cholesterol, lovastatin and Sandoz 58-035 had no effect on sitosterol absorption. The possibility has been demonstrated of using human small intestine organ culture and rat small intestine epithelial cell culture IRD-98 as models for studying cholesterol absorption.

Cholesterol uptake in the human intestine. Hypo- and hyperresponsiveness.

Cholesterol uptake was studied at the small intestine biopsies taken from patients without intestinal malfunction. Three distinct groups of patients were described: those with low (146 +/- 19) nmol/mm2 per 2 h), medium (455 +/- 18 nmol/mm2 per 2 h) and high (833 +/- 24 nmol/mm2 per 2 h) rates of cholesterol uptake. Positive correlation between cholesterol uptake and intestinal cholesterol synthesis was observed in the last two groups.

New model to study cholesterol uptake in the human intestine in vitro.

A new model to study cholesterol uptake in the human intestine in vitro is described. Human small intestine organ cultures were incubated with mixed micelles containing bile acid, phospholipid, and cholesterol or its nonabsorbable analogue, sitosterol; trace amounts of labeled cholesterol or sitosterol were added to the micelles. After incubation, the lipids were extracted from the cells and cholesterol and sitosterol uptake was evaluated. Specific cholesterol uptake was determined as a difference between cholesterol and sitosterol uptake. Cholesterol, but not sitosterol, uptake was time- and dose-dependent. Rapid and slow phases of cholesterol uptake were observed. Cholesterol uptake was also temperature-dependent. Removal of epithelial cells from human intestine explants reduced cholesterol, but not sitosterol, uptake. Inhibition of acyl CoA:cholesterol acyltransferase by Sandoz compound 58-035 and treatment with monensin reduced cholesterol uptake, but not sitosterol uptake, in a dose-dependent manner. In contrast, treatment of cultures with an inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, lovastatin, stimulated cholesterol, but not sitosterol, uptake in a dose-dependent manner; mevalonic acid reversed the effect of lovastatin. The presented model allows large-scale in vitro studies of different stages of cholesterol absorption in the human intestine.

Studies on the proteins involved in the interaction of high-density lipoprotein with isolated human small intestine epithelial cells.

Treatment of 125I-labelled high-density lipoprotein ([125I]HDL3) with monospecific polyclonal antibodies against apolipoproteins A-I and A-II resulted in a dose-dependent inhibition of the [125I]HDL3 binding to isolated human small intestine epithelial cells by 25% and 50%, respectively. Both antibodies also inhibited intracellular degradation of [125I]HDL3 by 80%. Treatment of enterocytes with polyclonal antibody against apolipoprotein A-I binding protein, a putative HDL receptor, inhibited both binding and degradation of [125I]HDL3 by these cells by 50%. Antibodies to apolipoprotein A-I, A-II and apo A-I-binding protein also inhibited [125I]HDL3 binding to cholesterol-loaded cells.

Ontogenetic regulation of apolipoprotein B mRNA editing during human and rat development in vivo.

The solubilization and delivery of lipids in plasma rely on both forms of apolipoprotein B (apo B): apo B-100 and apo B-48. Apo B-48 is the translational product of apo B-100 mRNA that undergoes peritranscriptional conversion of C----U, replacing codon CAA (glutamine 2,153) with the inframe stop codon (UAA). We examined mRNA editing activity in the human and the rat by reverse transcription-polymerase chain reaction primer-extension analysis of intestine and liver total RNA. In rat intestine the percentage of apo B transcripts that undergo editing increases dramatically the day before birth (from approximately 1% to 80%), whereas the rat liver acquires an adult level of editing activity during the third postnatal week (rising from approximately 8% to 30%), when weaning is completed, bile acid composition matures, and plasma thyroid hormone levels peak. In contrast to the rat, the human intestine acquires adult levels of apo B mRNA editing relatively early in fetal development, rising from 10% at 10 weeks to approximately 80% by the end of the second trimester. Our results establish that apo B mRNA editing is 1) developmentally regulated in a tissue- and species-specific manner; 2) fully developed prenatally in both human and rat intestine, suggesting a crucial role of apo B-48 in mammalian fetal adaptation to extrauterine life; and 3) acquired early in human fetal intestine, implying a potential role for apo B-48 in prenatal lipid metabolism.

Comparison of the effect of six compactin-related compounds on cholesterol synthesis in five human cell types.

We have investigated the effect of six compactin-related compounds--mevinolin, compactin, ML-236A, monacolin X, monacolin L and dihydromonacolin L--on cholesterol synthesis in human umbilical vein endothelial cells, human small intestine epithelial cells, human hepatoma cell line HEP G2, normal human skin fibroblasts and in skin fibroblasts from a patient with familial homozygous hypercholesterolemia. The inhibition of cholesterol synthesis was found to depend on both the cell type and the type of compound used. The most effective compounds were mevinolin and compactin. Monacolin X, monacolin L and ML-236A were less effective, and dihydromonacolin L was the least efficacious. Endothelial and epithelial cells were sensitive to very low concentrations of inhibitors (IC50 = 1.0-30 pg/mL), HEP G2 cells required higher concentrations (IC50 = 0.01-66 ng/mL) and fibroblasts needed even higher concentrations (IC50 = 0.1-200 ng/mL). Lactone and acid forms of the inhibitors were equally active. None of the inhibitors had any effect on either protein or fatty acid synthesis in any of the cell types studied. It can be concluded that different compactin-related compounds show a range of potencies as cholesterol synthesis inhibitors and a dose-dependent tissue-selectivity.

Both apolipoproteins B-48 and B-100 are synthesized and secreted by the human intestine.

Apolipoprotein B (apoB), an apolipoprotein associated with very low density lipoproteins and the atherogenic low density lipoproteins (LDL), directs the metabolism of lipoprotein particles in plasma by interacting with the LDL receptor. Utilizing human intestinal biopsy organ cultures, we have studied the synthesis of intestinal apoB in man. Intestinal organ cultures from normal adults (n = 6) were incubated in the presence of protease inhibitors in media supplemented with [35S]methionine. Media from these cultures were evaluated by sequential NaDodSO4 polyacrylamide gel electrophoresis, radioautography, and Western blot analyses, and intestinal biopsies were studied using immunohistochemistry. The relative abundance of apoB-100 and apoB-48 mRNA was assessed using reverse transcriptase-polymerase chain reaction followed by primer extension. Although apoB-48 was the principal isoprotein that was newly synthesized by intestinal organ cultures, apoB-100 was also synthesized and secreted by human intestinal organ cultures with 16 +/- 3% of the intestinal apoB mRNA coding for apoB-100. These results establish that apoB-100 is produced by the human intestine. The synthesis of the atherogenic apoB-100 by the intestine has pathophysiologic implications for the development of diet-induced atherosclerosis.

Inhibition of cholesterol synthesis and esterification regulates high density lipoprotein interaction with isolated epithelial cells of human small intestine.

The effect of two inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lovastatin and monacolin L, and an inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), Sandoz compound 58-035, on the interaction of 125I-labeled high density lipoprotein-3 (HDL3) with isolated human enterocytes was studied. Both HMG-CoA reductase inhibitors inhibited cholesterol synthesis and 125I-labeled HDL3 binding and degradation by enterocytes; a strong correlation between changes in cholesterol synthesis and interaction of 125I-labeled HDL3 with cells was observed. Lovastatin caused reduction of the apparent number of 125I-labeled HDL3 binding sites without affecting the binding affinity. No changes of cell cholesterol content were observed after incubation of cells with lovastatin. Mevalonic acid reversed the effect of lovastatin on 125I-labeled HDL3 binding. Lovastatin blocked up-regulation of the HDL receptor in response to loading of cells with nonlipoprotein cholesterol and modified cholesterol-induced changes of 125I-labeled HDL3 degradation. Lovastatin also reduced HDL-mediated efflux of endogenously synthesized cholesterol from enterocytes. The ACAT inhibitor caused a modest increase of 125I-labeled HDL3 binding to enterocytes and significantly decreased its degradation; both effects correlated with inhibition of cholesteryl ester synthesis. The results allow us to assume that the intracellular free cholesterol pool may play a key role in regulation of the HDL receptor.

Inhibition of cholesterol synthesis by lovastatin tested on six human cell types in vitro.

Lovastatin (mevinolin) caused a strong and dose-dependent inhibition of cholesterol synthesis in six types of cultured human cells. Fifty percent inhibition of cholesterol synthesis in human enterocytes was observed at a lovastatin concentration of about 0.004 ng/ml and in other cells at a lovastatin concentration of about 0.03 ng/ml. At lovastatin concentrations between 1.0 and 100.0 ng/ml, a moderate tissue selectivity of lovastatin action was noted. At optimal concentrations, lovastatin inhibited cholesterol synthesis in hepatocytes by 98%, in normal and LDL-receptor negative fibroblasts, arterial smooth muscle cells and hepatoma G-2 cells by about 90%, and in enterocytes by 75%. In rat enterocytes lovastatin inhibited cholesterol synthesis by only 60%.

[The effect of temperature and Ca2+ on the interaction of high-density lipoproteins with epithelial cells in the human small intestine]. / Vliianie temperatury i Ca2+ na vzaimodeistvie lipoproteidov vysokoi plotnosti s kletkami épiteliia tonkogo kishechnika cheloveka.

The effect of temperature and Ca2+ ions on the interaction of high-density lipoprotein (HDL3) with human enterocytes was studied. It was shown that Kd measured at 4 degrees C is similar to that at 37 degrees C. Maximal number of binding sites at 37 degrees C is 15-fold times higher than that at 4 degrees C. EDTA (10 mM) and CaCl2 (0.5-5 mM) did not affect binding and uptake of HDL3 by human enterocytes. The obtained results indicate that HDL3-binding with these cells depends on temperature and does not depend on Ca2+ ions.

[A method for obtaining a long-lived intestinal tissue culture]. / K metodike polucheniia dolgozhivushchei intestinal'noi kul'tury tkani.

The interest in the studies of the intestines using the method of tissue organ culture has considerably grown in recent years. It can be explained by the great possibilities of obtaining unique data about the state of intestines in normal and pathological condition, e.g. malabsorption syndrome. The paper deals with the method modified by the authors to obtain long-living (24 hours) intestinal tissue organ culture. The investigations used bioptic sections which were obtained by jejunoscopy with spot biopsy of children suffering from intestinal malabsorption. The viability of the explants was proved by histological and histochemical tests. The promise held by the methods is emphasized.

Binding of partially reassembled high-density lipoprotein to isolated human small intestine epithelial cells. Effect of lipid composition.

The binding of human high-density lipoprotein (HDL3), apolipoprotein A-I (apoA-I) and recombinants of apoA-I with cholesterol and/or dimyristoylphosphatidylcholine (DMPC) to the HDL receptor on isolated human small intestine epithelial cells was studied. ApoA-I competed for 125I-labelled HDL3 binding sites less effectively than HDL3, and a lower amount of 125I-labelled apoA-I than 125I-HDL3 was bound to cells. The apoA-I/DMPC recombinant competed for 125I-HDL3 binding sites nearly as well as HDL3, and 125I-apoA-I/DMPC recombinant bound to cells with at least the same efficiency as 125I-HDL3. The apoA-I/DMPC/cholesterol recombinant failed to compete for 125I-HDL3 binding sites, and the 125I-apoA-I/DMPC/cholesterol complex binding to cells was several-fold lower than that of other particles. All particles bound to cells with similar dissociation constants. Tetranitromethane-modified HDL3 failed to bind to high-affinity specific binding sites and compete with 125I-HDL3 for binding. The results obtained make it possible to assume that, while apoA-I may be a determinant of the HDL receptor, the lipid composition of the lipoprotein may affect its interaction with the receptor.

Cholesterol synthesis in the small intestine of patients with malabsorption syndrome.

Cholesterol synthesis was studied in intestinal biopsies obtained from children with different forms of malabsorption syndrome. It was demonstrated that intestinal cholesterol synthesis is enhanced in celiac disease and several other forms of malabsorption syndrome. Mevinolin inhibited intestinal cholesterol synthesis in all groups of patients. No correlation was found between cholesterol synthesis and age and sex of the patients, clinical manifestation of the disease and plasma cholesterol level.

Cholesterol regulates high-density lipoprotein interaction with isolated epithelial cells of human small intestine.

The effect of cholesterol on the interaction of high-density lipoprotein (HDL) with isolated human small intestine epithelial cells (enterocytes) was studied. 125I-labeled HDL3 binding by these cells was enhanced in response to cholesterol loading of the cells in a time- and dose- dependent manner. Preincubation of the cells with cholesterol led to the enhancement both of the number of binding sites and the binding affinity. The enhancement of binding correlated with the cellular cholesterol content. Cycloheximide (0.5 mM) inhibited uptake of cholesterol by enterocytes and blocked its effect on 125I-labeled HDL3 binding. The effect of cholesterol on 125I-labeled HDL3 degradation had a double-phase character. At concentrations 10-20 micrograms/ml, the degradation rate was rapidly elevated, but further increase in cholesterol concentration led to a fall in the degradation rate. Incubation of enterocytes with HDL3 resulted in the efflux of cholesterol from cells and its incorporation into HDL3. The results obtained make it possible to assume that binding and degradation of 125I-labeled HDL3 by human enterocytes are independently regulated by the cell total cholesterol content. Binding of HDL by enterocytes may result both in the degradation of HDL and cholesterol efflux from cells.