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Biomedical subjects

R Fumagalli

Publications and source records attributed to R Fumagalli.

At least 19 recordsLinked to original sources
PubMed1992

Simvastatin, an inhibitor of cholesterol biosynthesis, shows a synergistic effect with N,N'-bis(2-chloroethyl)-N-nitrosourea and beta-interferon on human glioma cells.

The effect of simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on human glioma cell growth was investigated. When incubated with simvastatin, cell proliferation decreased in a concentration-dependent fashion, as measured by cell number and [3H]-thymidine incorporation into DNA (concentration producing 50% inhibition, 60 nM). The effect was detectable 12 h after cells were exposed to the drug and persisted for 2 days. Addition of mevalonate to cells exposed effect of simvastatin in combination with beta-interferon and N,N'-bis(2-chloroethyl)-N-nitrosourea, both antitumoral drugs, was also evaluated by cell growth inhibition assay. The concentration producing 50% inhibition for each of these drugs was 650 units/ml and 50 nM, respectively. Subliminal concentrations of beta-interferon or N,N'-bis(2-chloroethyl)-N-nitrosourea were incubated together with 1 nM simvastatin. The data were analyzed with the aid of an isobologram using the concept of an envelope of additivity. Simultaneous cell exposure to simvastatin with either N,N'-bis(2-chloroethyl)-N-nitrosourea or beta-interferon produced a strong synergistic inhibitory effect on cell proliferation. These data provide in vitro support for the possibility that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, utilized as plasma cholesterol-lowering agents, could potentiate the effect of antiblastic drugs on tumor growth.

Anticholesteremic Agents↗
PubMed1992

Characterization of a family with moderate hypercholesterolemia and binding defective low density lipoprotein.

Familial defective apolipoprotein B-100 (FDB) is a genetic disorder presenting with hypercholesterolemia and abnormal low density lipoprotein (LDL) that binds poorly to LDL receptors. This disease appears to be caused by a mutation in the apo B gene. In the present study thirteen members of a family with moderate hypercholesterolemia (250-350 mg/dl) were investigated. Biochemical studies on cultured skin fibroblasts ruled out classical familial hypercholesterolemia (receptor deficiency). LDL from nine affected members displayed, in an "in vitro" cell binding assay, a reduced affinity (2.5 fold) for the receptor, and had normal electrophoretic mobility, size and chemical composition. Lp(a) levels in family members were comparable to those present in normolipidemics and lower than those observed in primary hypercholesterolemia. The disorder is transmitted over three generations as an autosomal codominant trait and all the affected members are heterozygotes and hypercholesterolemic.

Adolescent↗
PubMed1992

Ability of the LDL receptor from several animal species to recognize the human apo B binding domain: studies with LDL from familial defective apo B-100.

To verify whether the LDL receptors from different animal species recognize the binding domain of human apo B-100 we studied the interaction of LDL from control and familial binding defective apo B-100 (FDB) with cultured cells. Human, monkey, bovine, guinea pig and rabbit LDL receptors distinguish between normal and binding defective LDL with a displacement ratio (defective/normal) of 3.3, 2.6, 3.4, 3.1 and 2.0, respectively. Guinea pig and rabbit receptors, however, showed affinities 2-3-fold lower than the human receptor. Hamster, rat and mouse cells failed to differentiate between normal and FDB LDL with a ratio of 1.2, 0.8, and 1.4; the apparent affinities were 4-8 times lower than that of the human receptor. The data from the latter species suggest that the LDL receptor recognizes an area of human apo B different from the human receptor binding domain. The ability of antibody Mb47 to inhibit the binding of human LDL to human, rabbit and guinea pig but not to mouse cells further stresses this concept. Moreover, in 17 alpha-ethinyl estradiol-treated rats the rate of disappearance from plasma of FDB and control 125I-labelled LDL was identical, thus confirming the in vitro observations. These data suggest that the binding domain of the LDL receptor is functionally conserved in man, monkey, cow, rabbit and guinea pig, but is quite distinct in rat, mouse and hamster.

Animals↗
PubMed1992

Low density lipoprotein receptor activity is modulated by soybean globulins in cell culture.

The effects of major storage globulins from soybean on cholesterol homeostasis were investigated in vitro and in vivo systems. The low density lipoprotein (LDL) uptake and degradation was studied both in human skin fibroblasts (HSF) and in a human hepatoma cell line (Hep G2). In Hep G2 cells a dose-dependent increase of both uptake and degradation of 125I-LDL was induced by the 7S globulin, whereas the 11S globulin exerted a lesser effect that was not dose-related. In HSF cells the 11S globulin increased the uptake of 125I-LDL to a greater extent than did 7S globulin; in this cell line, LDL degradation was not stimulated by either of the globulins. Rats fed a casein-cholesterol diet were treated daily with the 11S or 7S globulins for 2 wk. The administration of soybean globulins significantly reduced cholesterolemia (-35 and -34% with 7S and 11S globulins, respectively, vs. controls). Liver membrane preparations from the casein-cholesterol-fed rats showed a nonsignificant increase in the maximal binding of labeled cholesterol-rich lipoprotein fraction (beta-VLDL) to high affinity receptors.

Animals↗
PubMed1992

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor.

Antibodies, Monoclonal↗
PubMed1992

Defective catabolism of oxidized LDL by J774 murine macrophages.

In J774 murine macrophages, chemically oxidized LDL (OxLDL) and biologically oxidized LDL (BioOxLDL) have similar metabolic fates, characterized by a relatively poor degradation when compared with acetylated LDL (AcLDL), and a modest ability to activate acyl-CoA:cholesterol acyltransferase (ACAT) (850 and 754 pmol [14C]oleate/mg cell protein in OxLDL- and BioOxLDL-incubated cells, versus 425 and 7070 pmol [14C]cholesteryl oleate/mg cell protein in control and AcLDL-incubated cells) with a massive increase of cellular free cholesterol. Therefore, OxLDL were used to investigate the cellular processing of oxidatively modified LDL. Binding and fluorescence microscopy studies demonstrated that OxLDL are effectively bound and internalized by macrophages and accumulate in organelles with density properties similar to those of endo/lysosomes. Although the overall metabolism of OxLDL is modestly affected by 100 microM chloroquine, owing to the poor cellular degradation of the substrate, the drug can further depress OxLDL degradation, indicating that this process takes place in an acidic compartment. Failure to detect products of extensive degradation of OxLDL in the medium is due to their relative resistance to enzymatic hydrolysis, as demonstrated also by in vitro experiments with partially purified lysosomal enzymes, rather than to the intracellular accumulation of degradation products (degraded intracellular protein is, at most, 8.5% of total). This sluggish degradation process is not due to a cytotoxic effect since OxLDL do not affect the intracellular processing of other ligands like AcLDL or IgG. The accumulation of OxLDL-derived products within macrophages may elicit cellular responses, the relevance of which in the atherosclerotic process remains to be addressed.

Animals↗
PubMed1991

Cholesterol stimulation of HDL binding to human endothelial cells EAhy 926 and skin fibroblasts: evidence for a mechanism independent of cellular metabolism.

The properties of the HDL binding site on the permanent human cell line EAhy 926 were studied. This cell line presents with highly differentiated functions of vascular endothelium. EAhy 926 cells possess HDL3 saturable binding sites with a Kd of about 20 micrograms/ml, which were up-regulated by cholesterol and were pronase- and EDTA-insensitive. Furthermore, HDL3 promoted cholesterol efflux from EAhy 926 cells in a dose-dependent manner. Thus, the HDL-binding site in EAhy 926 cells is similar to that present in fibroblasts, smooth muscle cells and endothelial cells. Up-regulation of HDL binding by cholesterol did not require de novo synthesis of HDL 'receptor' protein, as shown by the lack of effect of cycloheximide and alpha-amanitin and also occurred in fixed, non-living cells. Similar results were obtained using human skin fibroblasts. From these data we conclude that: (a) EAhy 926 cells are a good model for studying the HDL interaction with endothelial cells; (b) a mechanism independent of cellular metabolism is involved in the cholesterol-mediated up-regulation of HDL binding sites in EAhy 926 cells and human skin fibroblasts.

Binding, Competitive↗
PubMed1991

Influence of trapidil and derivatives on cholesterol synthesis and esterification in cultured cells.

The effect of trapidil (Rocornal) and its derivatives AR 12456 and AR 12463 on endogenous cholesterol synthesis and on cholesterol esterification rate was studied in human skin fibroblasts (HSF), in human hepatoma cell line Hep G2 and in primary culture of peritoneal macrophages from mouse (PMM). The cholesterol esterification rate was not influenced by the drugs in the tested cell lines. The incorporation of [14C]acetate into cholesterol in HSF was inhibited by AR 12463 and AR 12456, but not by trapidil. The inhibitory potency of AR 12456 in HSF was enhanced after preincubation of the drug with Hep G2 and removal of the medium to HSF, suggesting that the formed metabolite(s) are more potent inhibitors than the parent substance. The metabolite(s) formed seem(s) to influence the first steps in the endogenous formation of cholesterol, because the incorporation of [14C]mevalonate into cholesterol was not significantly inhibited. These findings suggest that the demonstrated inhibition of the endogenous cholesterol synthesis by AR 12456, especially after transformation into a probably more active substance(s), together with the recently described enhanced expression of LDL receptors in Hep G2 cells may partially explain the hypocholesterolaemic activity of AR 12456.

Acetates↗
PubMed1991

Simvastatin but not pravastatin inhibits the proliferation of rat aorta myocytes.

The in vitro effect of simvastatin and pravastatin, two competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, on the proliferation of rat aortic smooth muscle cells was investigated. Simvastatin, but not pravastatin, inhibited the replication of arterial myocytes in concentrations ranging between 0.01 microM and 10 microM. The inhibition, evaluated as cell number and nuclear incorporation of [3H]thymidine, was dose-dependent and completely prevented by addition of mevalonate (100 microM), confirming the role of mevalonate or its products in regulating cell division and growth. The present results provide evidence that simvastatin, in addition to its effect on cholesterol biosynthesis, interferes in vitro with other processes involved in atherogenesis.

Animals↗
PubMed1991

Familial defective apo B-100, characterization of an Italian family.

Familial defective apolipoprotein (apo) B-100 is a genetic disorder presenting with hypercholesterolaemia and abnormal low-density lipoprotein (LDL) that binds poorly to LDL receptors. This disease appears to be caused by a mutation in the apo B-100 gene. In the present study thirteen members of a family with moderate hypercholesterolaemia (250-350 mg dl-1) were investigated. Biochemical studies on cultured skin fibroblasts ruled out classical familial hypercholesterolaemia (FH, receptor deficiency). We then studied the interaction between LDL and their receptors by an in vitro cell binding assay. LDL from nine affected members displayed a reduced affinity (2.5-fold) for the receptor, and were less effective than LDL from control and unaffected members in suppressing LDL receptor expression and in stimulating cholesterol esterification. LDL from the affected members had normal electrophoretic mobility, size and chemical composition. Partial delipidation did not modify the LDL binding defect. The disorder is transmitted over three generations as an autosomal codominant trait and all the affected members are heterozygotes and hypercholesterolaemics. Analysis of DNA from family members showed a point mutation leading to an Arg to Gln substitution at amino acid 3500 of the mature protein that segregated with hypercholesterolaemia and LDL defective binding. We conclude that this family is affected by familial defective apolipoprotein B-100 (FDB).

Adolescent↗
PubMed1991

Calcium antagonists and cholesteryl ester metabolism in macrophages.

Experimental data indicate that calcium antagonists modify cellular lipid metabolism in the arterial wall as part of their antiatherosclerotic action observed in animal models. In the present study, we investigated the effect of verapamil, nifedipine, and lacidipine (a new dihydropyridine derivative) on cholesteryl ester metabolism in cultured mouse peritoneal macrophages (MPMs). Cholesteryl esters are formed in the cell via acyl-CoA:cholesterol acyltransferase (ACAT), which senses free cholesterol supplied by lysosomal hydrolysis of lipoprotein cholesterol ester. Verapamil inhibited up to 99% the ability of acetyl-low-density lipoprotein (acLDL) to stimulate cholesterol esterification in macrophages, but was less effective in 25-hydroxycholesterol-stimulated MPMs and in cholesterol-loaded cells after acLDL removal. Cells incubated with [3H]cholesterol ester-acLDL and verapamil showed a reduction in the cholesterol/cholesteryl ester ratio. In the same experimental conditions, nifedipine displays minor or no effects on cholesterol esterification and in the cholesterol/cholesteryl ester ratio. On the contrary, the nifedipine-like lacidipine was active in inhibiting cholesterol esterification in macrophages elicited by acLDL. Our data indicate that calcium antagonists of different structure, even within the same class, may have various effects on cholesterol esterification in macrophages in culture.

Animals↗
PubMed1991

Increased affinity of LDL for their receptors after acipimox treatment in hypertriglyceridemia.

The regulation of cellular LDL metabolism by hypertriglyceridemic LDL was tested before and after treatment with acipimox, a nicotinic acid derivative, in 11 type IV hyperlipidemic patients. Large, less dense LDL particles were found in plasma after acipimox treatment. HDL subfractions were only slightly modified, with an increase of dense, cholesteryl ester-enriched and triglyceride-poor HDL3 particles. The LDL (B, E) receptor activity in human skin fibroblasts of LDL isolated before and after treatment was also evaluated. Hypertriglyceridemic LDL proved rather inefficient in regulating receptor activity, with a close to 30% lower capacity than normal LDL to inhibit receptor-mediated uptake and degradation of 125I-LDL. This abnormality was fully corrected after acipimox. The reported findings indicate that acipimox treatment in type IV patients can normalize the defective interaction of hypertriglyceridemic LDL with the LDL (B, E) receptor.

Adult↗