P. Roma

Experimental hypothyroidism modulates the expression of the low density lipoprotein receptor by the liver

The effect of experimental hypothyroidism on the catabolism of plasma lipoproteins and on the expression of low density lipoprotein receptors by the liver was investigated in rats made hypothyroid by surgery. The animals developed mild hypercholesterolemia, mainly due to an increase of plasma low density lipoprotein, while other lipoprotein classes were only marginally affected. Kinetic studies using [125I]LDL indicated that a decreased fractional catabolic rate of the lipoprotein was responsible for this finding in agreement with the in vitro observation of a reduced binding of lipoproteins to liver membranes from hypothyroid rats and with the demonstration, by ligand blotting analysis, of a decreased expression of lipoprotein receptors in liver membranes. These data suggest that hypothyroidism affects lipoprotein distribution also by decreasing the catabolism of low density lipoproteins by the liver.

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.

Oxidized LDL increase free cholesterol and fail to stimulate cholesterol esterification in murine macrophages

Oxidatively modified low density lipoproteins (Ox-LDL) may be involved in determining the formation of foam cells by inducing cellular cholesteryl ester accumulation. We studied the effect of copper oxidized LDL (Ox-LDL) on cholesterol accumulation and esterification in murine macrophages. Ox-LDL (44 micrograms/ml of lipoprotein cholesterol) increased the total cholesterol content of the cells from 29 to 69 micrograms/mg cell protein. Free cholesterol accounted for 85% of this increase. Acetyl LDL (Ac-LDL) (38 micrograms/ml of lipoprotein cholesterol), raised total cellular cholesterol content to a similar extent (76 micrograms/mg cell protein), however only 25% of the accumulated cholesterol was unesterified. When ACAT activity was determined after incubation of J774 cell with Ox- or Ac-LDL, Ox-LDL were 12 times less effective than Ac-LDL in stimulating cholesteryl ester formation. This was not due to an inhibition of ACAT by Ox-LDL since these lipoproteins failed to inhibit pre activated enzyme in cholesteryl ester-loaded macrophages. The uptake of 125I-Ox-LDL: was 175% that of 125I-Ac-LDL, while degradation was only 20%. All together these data suggest an altered intracellular processing of Ox-LDL, which may be responsible for free cholesterol accumulation.

Protection of low-density lipoprotein from oxidation by 3,4-dihydroxyphenylethanol

be given under such circumstances?’ If needed psychiatric care is not provided, what will happen to the untreated prisoner?’ Alternatively, if treatment is given, albeit involuntarily, to restore competence for execution, unsettled constitutional, in addition to ethical, issues may be raised. The main mission of psychiatrists, and physicians generally, is to serve as healers, rather than as cogs in the machine of the state.’ The issue of treating patients incompetent for execution places psychiatrists in a seeming lose-lose situation: it is difficult to abstain from treating the needy patient, yet the eventual result of successful treatment is death. There remains a salient need for guidelines that may better enable psychiatrists to navigate through the choppy, unsettled waters of participation in capital punishment.

Evidence for the presence of 7-hydroperoxycholest-5-en-3β-ol in oxidized human LDL

Low density lipoprotein (LDL) cholesterol is known to be oxidized both in vitro and in vivo giving rise to oxygenated sterols. Conflicting results, however, have been reported concerning both the nature and the relative concentrations of these compounds in oxidized human LDL. We examined the extracts obtained from Cu(2+)-oxidized LDL. Thin layer chromatography analysis showed that the sterol mixture became more complex with reaction time. Analysis of the components by thin layer chromatography and mass spectrometry allowed to establish that 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha OOH and beta OOH) are largely prevalent among the oxysterols at early times of oxidation. These hydroperoxy derivatives have not been previously identified in oxidized LDL. The concentration of 7-hydroperoxycholest-5-en-3 beta-ol decreased with oxidation time with a concomitant increase of cholest-5-en-3 beta, 7 alpha-diol (7 alpha OH), cholest-5-en-3 beta, 7 beta-diol (7 beta OH), cholesta-3,5-dien-7-one (CD) and cholest-5-en-3 beta-ol-7-one (7CO). After 24 h of oxidation a minor component of the LDL sterols was cholestan-3 beta-ol-5,6-oxide (EP).

Stress proteins and atherosclerosis

Several cytotoxic stimuli of a different nature are involved in the complex etiology of atherosclerosis. Cells of the vasculature may potentially cope with the presence of these stressors through the increased synthesis of stress proteins (or heat shock proteins, hsps), an ubiquitous and conserved defense response. Evidence exists that the expression of two stress proteins of intermediate molecular weight, hsp60 and hsp70, is higher at sites of atherosclerotic lesions than it is in normal tissue. The role of hsps in atherosclerosis is controversial. While hsp70 is likely to be involved in cytoprotection, hsp60 is probably acting as an autoantigen, and may trigger both cell-mediated and antibody-mediated immune responses.

Human Endothelial Cells Exposed to Oxidized LDL Express hsp70 Only When Proliferating

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, is cytotoxic and triggers the expression of various heat shock proteins (hsps), among which is hsp70, in cultured animal and human cells. hsps constitutively act as molecular chaperones and in situations of stress protect other cellular proteins from potential denaturation caused by cytotoxic stimuli. The sensitivity of endothelial cells to OxLDL toxicity and accordingly the level of hsp70 expression depend on cell density. While confluent cells were relatively resistant to OxLDL toxicity and were not induced to express hsp70 when challenged with the lipoprotein (up to 800 micrograms/mL), sparse cells exhibited a concentration- and time-dependent expression of inducible hsp70, which increased up to fivefold to sixfold in unchallenged cells. Neither the activity of receptors recognizing OxLDL nor potentially protective cell products affected the stress response. Rather, we demonstrated that cell proliferation, which is high for sparse cultures and wound-healing monolayers, is responsible for these observations. We also demonstrated that the lipid moiety of OxLDL essentially accounts for the hsp-inducing effect of the lipoprotein. OxLDL has been detected in atherosclerotic lesions, which also show an increase of immunoreactive hsp72/73. We speculate that, in vivo, rapidly growing cells, such as those of lesion-prone areas, are more sensitive to the toxicity of OxLDL than are quiescent cells and that an increased expression of hsp70 may allow proliferating cells an increased chance of survival.

Oxidized LDL induce hsp70 expression in human smooth muscle cells

Heat shock protein 70 (hsp70) has been detected in atherosclerotic lesions, in which endothelial cells and smooth muscle cells are involved. In a previous report we showed that Ox‐LDL, a causal factor in atherosclerosis, could induce hsp70 expression in cultured human endothelial cells [Zhu et al. B.B.R.C 1994, 200: 389]. Here, with immunofluorescence and immunoblotting techniques, we show that Ox‐LDL are capable of inducing hsp70 expression also in human smooth muscle cells, and that this induction is dependent on cell density and on the concentration of Ox‐LDL. The induced expression of hsp70 was higher in human umbilical vein smooth muscle cells than in a human smooth muscle cell line. Conversely, Ox‐LDL was cytotoxic to both types of cells, more so to the human smooth muscle cell line. These observations indicate that Ox‐LDL may be a stress responsible for hsp70 expression in atherosclerotic plaques and the presence of hsp70 in plaques may be a useful marker for continuous oxidative damage in the arterial wall.

Plasma lipids, lipoproteins and apoproteins in a case of apo C-II deficiency

A new case of apo C-II deficiency is described. The patient had plasma triglyceride levels ranging from 10.2-30.5 mmol/l. Apo C-II deficiency was confirmed by gel electrophoresis, isoelectric focusing and immunochemistry. In this patient plasma lipoproteins were mainly chylomicrons and very low density lipoproteins, LDL and HDL levels being very low. Infusion of normal plasma effectively reduced plasma triglycerides and enhanced low density and high density lipoproteins cholesterol levels. These data suggest that in vivo a precursor-product relationship exists between triglyceride rich lipoproteins and LDL and HDL, and further stress the role of the lipoprotein lipase-apo C-II system in modulating these metabolic interconversions.

Bile lipid composition and haemostatic variables in a case of high density lipoprotein deficiency (Tangier disease)

Abstract. A 62‐year‐old man with clinical and biochemical findings consistent with homozygous Tangier disease is presented. Widespread atherosclerosis was present. Bile lipid analysis showed a low molar percentage of cholesterol with a low saturation index. The data suggest that high density lipoprotein cholesterol may act as a preferential precursor of bilary cholesterol. Coagulation and platelet studies indicated that the patients platelets were hyper‐responsive to aggregating agents and produced an increased amount of thromboxane B2. A platelet storage pool deficiency was also found.