Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects

The profoundly elevated concentrations of low-density lipoproteins (LDL) present in homozygous familial hypercholesterolemia lead to symptomatic cardiovascular disease and death by early adulthood. Studies conducted in nonhepatic tissues demonstrated defective cellular recognition and metabolism of LDL in these patients. Since mammalian liver removes at least half of the LDL in the circulation, the metabolism of LDL by cultured hepatocytes isolated from familial hypercholesterolemic homozygotes was compared to hepatcytes from normal individuals. Fibroblast studies demonstrated that the familial hypercholesterolemic subjects studied were LDL receptor-negative (less than 1% normal receptor activity) and LDL receptor-de fective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL (Bmax = 2.2 μg LDL/mg cell protein). Preincubation of normal hepatocytes with 200 μg/ml LDL reduced binding and internalization by approx. 40%. In contrast, 125I-labeled LDL binding and internalization by receptor-negative familial hypercholesterolemic hepatocytes was unaffected by cholesterol loading and considerably lower than normal. This residual LDL uptake could not be ascribed to fluid phase endocytosis as determined by [14C]sucrose uptake. The residual LDL binding by familial hypercholesterolemia hepatocytes led to a small increase in hepatocyte cholesterol content which was relatively ineffective in reducing hepatocyte 3-hydroxy-3-methylglutaryl-CoA reductase activity. Receptordefective familial hypercholesterolemia hepatocytes retained some degree of regulatable 125I-labeled LDL uptake, but LDL uptake did not lead to normal hepatocyte cholesterol content or 3-hydroxy-3-methylglutaryl-CoA reductase activity. These combined results indicate that the LDL receptor abnormality present in familial hypercholesterolemia fibroblasts reflects deranged hepatocyte LDL recognition and metabolism. In addition, a low-affinity, nonsaturable uptake process for LDL is present in human liver which does not efficiently modulate hepatocyte cholesterol content or synthesis. © 1986

Modification of Low Density Lipoprotein and the Effects of Modified Low Density Lipoproteins on the Endocytic and Secretory Repertoire of the Macrophage

This study examines two aspects of the complex interaction between modified low density lipoprotein (LDL) and macrophages in the initiation and development of the atherosclerotic lesion. The first of these concerns the observed biological modification of LDL that leads to its subsequent enhanced uptake by macrophages, and the role of oxidation in that process. The second aspect of this thesis studies the effects of modified LDL on the functions of macrophages. Studies on the oxidation of LDL showed that LDL isolated from human plasma anticoagulated with EDTA (EDTA-LDL) was 4-fold more resistant to oxidation by reagent H2O2, as measured by the thiobarbituric acid (TBA) assay, than LDL prepared from plasma anticoagulated with citrate (CDP-LDL). The LDLsrequired 1-3 mM H2O2 for maximal oxidation by this assay, and ED50s were 1.7 x 10-3 M for EDTA-LDL and 4.5 x 10-4 M for CDP-LDL. Oxidation was enhanced 2.3-fold by Cu2+ ions. Rabbit endothelial cell line monolayers released three orders of magnitude less H2O2 than was required to oxidize LDL and failed to induce TBA reactivity in either EDTA-LDL or CDP-LDL after a 24-hour coincubation. However, this LDL was subsequently degraded by mouse macrophages more rapidly than untreated LDL. Freshly isolated human monocytes (2 x 106 cells per ml), with or without phorbol myristate acetate (100 ng/ml) to trigger the respiratory burst, did not oxidize LDL in the TBA assay, despite producing large amounts of reactive oxygen intermediates. EDTA-LDL, CDP-LDL, and acetoacetylated LDL failed to trigger H2O2 release from human monocytes or macrophages. These results separate oxidation of LDL as measured by TBA assay from the modification of LDL by a rabbit aortic endothelial cell line that leads to its subsequent enhanced degradation by macrophages. Mouse peritoneal macrophages were lipid-loaded by three regimens modeling loading through the scavenger receptor (Ac-LDL cells), by extracellular matrix-bound LDL (DS-LDL cells), and conditions of reduced cholesterol acceptors in the medium (LS/01 cells). Increased cholesterol levels in all three regimens were measured by cholesterol determination, Oil Red 0 staining of fixed cells, and extraction of Oil Red 0 from cells and determination of OD530. Lipid-laden cells were equal to control macrophages in binding and ingesting immunoglobulin-coated sheep erythrocytes, reflecting Fc-mediated endocytosis. Cellular proteins appeared largely unchanged, but alterations in the secreted protein profile were observed by metabolic labeling with 35s-methionine. DS-LDL loaded cells and LS/01 loaded cells showed increases in a 68 kD protein, and decreases in apo E secretion. Ac-LDL loaded cells showed significant enhancement in apo E secretion. The lipid-laden cells were compared to control cells for three secretory functions of macrophages that could be important in the atherosclerotic plaque. They were still capable of producing all secretory products examined, but the quantities of H2O2 and arachidonic acid metabolites are reduced in some cases, and plasminogen activator appeared to be increased. However, we have lipid-loaded the cells for only two days, and foam cells in vivo may be exposed to these conditions for months or years. The different loading regimens result in different changes, which we have separated, but in vivo the cells would be exposed to a complex mixture of native and biologically modified LDL, and LDL complexed glcosaminoglycans and with proteins of the extracellular matrix. It is premature to predict what changes in the macrophage\u27s large repertoire would occur in vivo

Distribution of exogenous 25-hydroxycholesterol in Hep G2 cells between two different pools

AbstractBinding of [26,27-3H]25-hydroxycholesterol (25HC) to human hepatoma Hep G2 cells was saturated within 120 min. Two intracellular pools of 25HC were identified in a pulse-chase experiment: (i) an exchangeable pool which was in dynamic equilibrium with 25HC in the medium (t1/2 of reversible exchange 15 min) and (ii) an unexchangeable pool which remained in cells during incubation in medium containing LPDS. 25HC from the exchangeable pool inhibits cholesterol biosynthesis, decreases the HMG CoA reductase mRNA level and stimulates cholesterol acylation. 25HC from the unexchangeable pool was partially bound to cytosolic proteins and apparently utilized for metabolic transformation. Incubation of Hep G2 cells with [26,27-3H]25HC in the presence of a 30-fold molar excess of 3β-hydroxy-5α-cholest-8(14)-en-15-one was found to cause (i) 2-fold decrease in the binding of [26,27-3H]25HC to cytosolic proteins (sedimentation constant of radioactive complex was 4–5 S) and (ii) the 35% inhibition of 25HC transformation to polar metabolites

Impaired ATP-binding cassette transporter A1-mediated sterol efflux from oxidized LDL-loaded macrophages

AbstractWe investigated the interaction of oxidized low density lipoprotein (OxLDL) with the ATP-binding cassette A1 (ABCA1) pathway in J774 macrophages. Cellular efflux to apolipoprotein AI (apo-AI) of OxLDL-derived cholesterol was lower than efflux of cholesterol derived from acetylated low density lipoprotein (AcLDL). ABCA1 upregulation by 8-(4-chlorophenylthio)adenosine 3′:5′-cyclic monophosphate (cpt-cAMP) or 22 (R)-hydroxycholesterol (22-OH) and 9-cis retinoic acid (9cRA) increased the efflux to apo-AI of cellular sterols derived from AcLDL, but not of those from OxLDL. AcLDL, but not OxLDL, induced ABCA1 protein content and activity in J774. However, OxLDL did not influence J774 ABCA1 upregulation by cpt-cAMP or 22-OH/9cRA. We conclude that sterols released to cells by OxLDL are available neither as substrate nor as modulator of ABCA1

Environmental contaminants exposure and preterm birth: a systematic review

Preterm birth is an obstetric condition associated with a high risk of infant mortality and morbidities in both the neonatal period and later in life, which has also a significant public health impact because it carries an important societal economic burden. As in many cases the etiology is unknown, it is important to identify environmental factors that may be involved in the occurrence of this condition. In this review, we report all the studies published in PubMed and Scopus databases from January 1992 to January 2019, accessible as full-text articles, written in English, including clinical studies, original studies, and reviews. We excluded articles not written in English, duplicates, considering inappropriate populations and/or exposures or irrelevant outcomes and patients with known risk factors for preterm birth (PTB). The aim of this article is to identify and summarize the studies that examine environmental toxicants exposure associated with preterm birth. This knowledge will strengthen the possibility to develop strategies to reduce the exposure to these toxicants and apply clinical measures for preterm birth prevention

The Role of the Reticuloendothelial System in LDL Metabolism

Prospective epidemiological studies show that raised plasma cholesterol is a major risk factor for the development of ischaemic heart disease. Recent evidence indicates that reducing plasma cholesterol concentration reduces risk from the disease. Cholesterol is transported in the plasma mainly by low density lipoproteins (LDL) and these particles also confer risk if present in high concentrations. It is important to determine the mechanisms by which LDL is removed from the plasma and catabolised by the tissues of the body. The arterial wall is especially important in this respect as it can accumulate excessive quantities of cholesterol. LDL catabolism is divided into two pathways: 1) LDL-receptor-dependent catabolism is now a well understood process by which a receptor recognises apolipoprotein B (the sole apolipoprotein moiety on LDL) and mediates the internalisation of the lipoprotein. It is then transported to the lysosome, degraded and the cholesterol released for cellular needs. The pathway is autoregulated by cellular requirements for the sterol. In most animals, including man, approximately one half of LDL catabolism occurs by this route. 2) LDL-receptor-independent catabolism is less well defined. Studies have indicated that the reticuloendothelial system plays a role in this scavenger pathway. The purpose of the present study is to increase understanding of the role played by the reticuloendothelial system (RE system) in LDL metabolism. However, because the RE system is a disseminated tissue it is not as easy to study as, for example, the liver which can even be removed from the animal intact. One approach to this problem is functionally to block the cells of the system by injection of material which will be phagocytoced by these macrophages. This was achieved using three agents; a) ethyl oleate emulsion, b) muramyl tripeptide incorporated into triolein emulsion and c) muramyl dipeptide conjugated to acetylated bovine serum albumin (for which the cells of the RE system have a receptor). The end result was the same regardless of the agent employed. The following summarises the essential conclusions of the thesis. 1) The RE system is important in lipoprotein catabolism. Blockage of the system causes an increase in plasma triglyceride and cholesterol levels. The raised total cholesterol is almost entirely accounted for by an increase in LDL cholesterol. 2) RE cells are important in the clearance of LDL from the plasma. Administration of RE cell activity inhibiting agents causes a decrease in the rate of the clearance of the lipoprotein. Kinetic analysis shows that this was due to a decrease in LDL-receptor-independent catabolism. 3) Lipoprotein catabolism by cultured macrophages is down regulated by RE system suppressants. Incubation of macrophages with these agents reduces the amount of LDL and B-VLDL catabolised by the cells. This supports the in vivo findings. 4) LDL is altered in the plasma of RE blocked animals. It has an increased density, an increased electrophoretic mobility (negative charge, an increased cholesterol to protein ratio and is sleared from the plasma more rapidly than normal LDL. Control LDL injected into RE suppressed animals assumes the characteristics of this abnormal lipoprotein. 5) Blocking the RE system of cholesterol fed animals appears to promote the atheromatous infiltration of their aortae. Plasma cholesterol levels in the RE blocked animals were not significantly different from those in cholesterol fed animals with functional RE systems but the overall negative charge of their LDL was greater. 6) It follows that the RE system is working to clear an abnormal form of LDL which may be formed by "ageing" in the circulation. Potentially this LDL is catabolised by atherosclerotic plaques leading to lesion advancement