Search PubMed⌕ Search

Biomedical subjects

D S Leake

Publications and source records attributed to D S Leake.

At least 55 records · Page 3Linked to original sources
PubMed1992

The oxidative modification of low density lipoprotein by human lymphocytes.

Oxidation of low density lipoprotein in atherosclerotic lesions may be involved in converting macrophages into lipid-laden foam cells. Lesions contain endothelial cells, smooth muscle cells, macrophages and lymphocytes. The first three types of cells have been shown previously to modify low density lipoprotein so as to increase its uptake by macrophages. We report here that lymphocytes from human blood are also capable of doing this. The modification process was an oxidative one because there was an increase in the thiobarbituric acid-reactive substances in media containing lymphocyte-modified low density lipoprotein and the modification could be inhibited by the antioxidants butylated hydroxytoluene and probucol. The lymphocyte-modified low density lipoprotein was taken up and degraded by macrophages by their scavenger receptor(s).

Animals↗
PubMed1990

The oxidative modification of low-density lipoproteins by macrophages.

1. Mouse resident peritoneal macrophages in culture modified human 125I-labelled low-density lipoprotein (LDL) to a form that other macrophages took up about 10 times as fast as unmodified LDL. The modified LDL was toxic to macrophages in the absence of serum. 2. There was a lag phase of about 4-6 h before the LDL was modified so that macrophages took it up faster. A similar time lag was observed when LDL was oxidized by 5 microM-CuSO4 in the absence of cells. 3. LDL modification was maximal when about 1.5 x 10(6) peritoneal cells were plated per 22.6 mm-diam. well. 4. Re-isolated macrophage-modified LDL was also taken up much faster by macrophages, indicating that the increased uptake was due to a change in the LDL particle itself. 5. Micromolar concentrations of iron were required for the modification of LDL by macrophages to take place. The nature of the other components in the culture medium was also important. Macrophages would modify LDL in Ham's F-10 medium but not in Dulbecco's modified Eagle's medium, even when iron was added to it. 6. The macrophage-modified LDL appeared to be taken up almost entirely via the acetyl-LDL receptor. 7. LDL modification by macrophages was inhibited partially by EDTA and desferrioxamine and completely by the general free radical scavengers butylated hydroxytoluene, vitamin E and nordihydroguaiaretic acid. It was also inhibited completely by low concentrations of foetal calf serum and by the anti-atherosclerotic drug probucol. It was not inhibited by the cyclo-oxygenase inhibitors acetylsalicylic acid and indomethacin. 8. Macrophages are a major cellular component of atherosclerotic lesions and the local oxidation of LDL by these cells may contribute to their conversion into cholesterol-laden foam cells in the arterial wall.

Animals↗
PubMed1990

Macrophage proteases can modify low density lipoproteins to increase their uptake by macrophages.

When low density lipoprotein (LDL) was incubated with sonicated macrophages at acidic pH, its protein moiety was partially degraded by cathepsins B and D. The reisolated LDL was taken up by intact macrophages up to about 20 times as fast as control LDL. LDL proteolysis and its enhanced uptake could be inhibited almost entirely by the selective protease inhibitors leupeptin and pepstatin. If macrophages in atherosclerotic lesions were to release acidic proteases (either by exocytosis or following cell death) and these were to modify LDL, this may help to explain why so much cholesteryl ester accumulates in these cells.

Animals↗
PubMed1990

Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages.

Low density lipoproteins (LDL) can be oxidatively modified in vitro by macrophages and certain other cell types so that macrophages will take them up much faster. This process may be important in the formation of cholesterol-laden foam cells derived from macrophages in atherosclerotic lesions. In this study, we have shown that certain flavonoids, plant constituents found in the diet, are potent inhibitors of the modification of 125I-labelled LDL by macrophages, with IC50 values in the micromolar range (e.g. morin and fisetin 1 microM; quercetin and gossypetin 2 microM). The potencies of individual flavonoids in inhibiting LDL modification did not correlate with their previously determined potencies as inhibitors of 5-lipoxygenase and cyclo-oxygenase. The modification of LDL by macrophages exhibits a lag period of about 4-6 hr before enhanced uptake is detected. During this time, there is a rapid depletion in its content of alpha-tocopherol (an endogenous antioxidant found in lipoproteins) followed by a large increase in the level of hydroperoxides. The flavonoids conserved the alpha-tocopherol content of LDL and delayed the onset of detectable lipid peroxidation. Flavonoids also inhibited the cell-free oxidation of LDL mediated by CuSO4. These findings raise the possibility that flavonoids may protect LDL against oxidation in atherosclerotic lesions and may therefore be natural anti-atherosclerotic components of the diet, although this will depend to a large extent on their pharmacokinetics.

Copper↗
PubMed1990

Alpha-tocopherol consumption during low-density-lipoprotein oxidation.

1. The kinetics of the depletion of alpha-tocopherol in human low-density lipoprotein (LDL) were measured during macrophage-mediated and cell-free oxidation. The formation of oxidatively modified, high-uptake species of LDL in these systems was not detectable until all of the endogenous alpha-tocopherol had been consumed. 2. Supplementation of the alpha-tocopherol content of LDL by loading in vivo extended the duration of the lag period during which no detectable oxidative modification occurred. 3. The addition of a flavonoid (morin) prevented both alpha-tocopherol consumption and oxidative modification of LDL. 4. The alpha-tocopherol contents of LDLs from a range of individual donors could not be used to predict their relative resistance to oxidation, indicating that other endogenous antioxidants may also be present, and quantitatively significant, in human LDL.

Animals↗
PubMed1990

Failure of platelet-derived growth factor plus insulin to stimulate sustained proliferation of Swiss 3T3 cells. Requirement for hydrocortisone, prostaglandin E1, lipoproteins, fibronectin and an unidentified component derived from serum.

Platelet-derived growth factor (PDGF) has been reported to be a potent mitogen for Swiss 3T3 cells made quiescent by growth to saturation density in high serum, and its activity is further potentiated by high levels of insulin, which alone have little effect. We show here that this is not the case for sparse 3T3 cells made quiescent by plating in low serum. Under these conditions, insulin alone is at least as effective as PDGF and frequently more so. Together, the response is no more than additive at best, and in many cases less than additive, the combined effect being no greater than for insulin alone. Instead, we find that optimal mitogenic stimulation requires the additional presence, besides PDGF and insulin, of hydrocortisone, prostaglandin E1 and an unidentified, non-dialysable component contained in serum treated with dithiothreitol (DTT) to inactivate endogenous growth factors. Interestingly, overnight pretreatment of the cells with hydrocortisone alone potentiates the subsequent response to PDGF + insulin, i.e. pretreatment induces a long-term memory that persists after the removal of the hydrocortisone from the medium. In short-term (24h) thymidine incorporation assays, the combination of PDGF, insulin, hydrocortisone, prostaglandin E1 and DTT-serum, is as effective as optimal levels of whole serum, but is unable to sustain longer-term proliferation (measured over 6 days). For this, high- and low-density lipoproteins, fibronectin and, to some extent epidermal growth factor (EGF), are also necessary.

Alprostadil↗
PubMed1989

The effect of macrophage stimulation on the uptake of acetylated low-density lipoproteins.

The rapid uptake of modified low-density lipoproteins (LDL) by macrophages in the arterial wall may lead to their conversion into lipid-laded foam cells in atherosclerotic lesions. We have therefore investigated the effects of macrophage stimulation on their rate of uptake of modified LDL. The uptake of 125I-labelled acetyl-LDL by mouse resident peritoneal macrophages was reduced by about 60-85% by zymosan (250 micrograms/ml), by 25-45% by lipopolysaccharide (0.1-1 mg/ml) and 50-60% by phorbol myristate acetate (100 nM). The inhibition was dose-dependent and was observed at the earliest times studied (about 1 h). Binding studies at 0 degrees C showed that all three stimulating agents decreased the number of cell-surface receptors for acetyl-LDL. If macrophages are stimulated in atherosclerotic lesions, this may therefore be beneficial in that it may decrease their numbers of receptors for modified LDL, although it may be harmful in other ways in that stimulated macrophages may release factors that damage the arterial wall.

Animals↗
PubMed1989

The effects of acetylated low-density lipoproteins on fluid-phase pinocytosis by macrophages.

A simple method has been set up to measure the rate of fluid-phase pinocytosis in resident mouse peritoneal macrophages in culture. The method uses 125I-labelled polyvinylpyrrolidone as a nondegradable marker of fluid-phase pinocytosis. The accumulation of 125I-labelled polyvinylpyrrolidone by the cells was directly proportional to its concentration in the culture medium up to at least 200 micrograms/ml. The estimates of the rate of fluid-phase pinocytosis were reproducible within each experiment (coefficient of variation 8.5%) but varied between individual experiments. Fluid-phase pinocytosis was undetectable at 4 degrees C and reduced greatly at 37 degrees C by metabolic inhibitors and 1 mM ZnSO4. High concentrations of human acetylated low-density lipoproteins, which are taken up rapidly by macrophages, decreased the rate of fluid-phase pinocytosis by up to about 70%. The inhibition was seen after only 2 h of incubation. Unmodified low-density lipoproteins, which are taken up only slowly by macrophages, did not usually inhibit fluid-phase pinocytosis (in fact, they sometimes increased it). Modified low-density lipoprotein uptake, leading to massive lipid accumulation in macrophages in the arterial wall, has been postulated to be involved in the pathogenesis of atherosclerosis. This study raises the possibility that the rate of fluid-phase pinocytosis in these lipid-laden arterial macrophages may be reduced.

Acetylation↗
PubMed1989

Macrophages possess both neutral and acidic protease activities toward low density lipoproteins.

Low density lipoproteins (LDL) have been strongly implicated in the pathogenesis of atherosclerosis. We have studied the proteolytic degradation of these lipoproteins by macrophages, which are a major cellular constituent of atherosclerotic lesions. Mouse peritoneal macrophages contained both an acidic and a less active but distinct neutral/alkaline protease activity toward human 125I-labelled LDL. The acidic activity had a pH optimum of 4.5 and the neutral/alkaline activity one of 8-8.5. The acidic activity started to plateau with increasing lipoprotein concentrations whereas the neutral activity was directly proportional to the lipoprotein concentration up to at least 150 micrograms of protein/ml. The acidic protease activity had a complex time course whereas the neutral activity was directly proportional to the time of incubation up to at least 48 h. Leupeptin (35 microM) and pepstatin (5 microM) inhibited the acidic activity by about 70% individually and almost entirely in combination, indicating that cathepsins B and D are important in the degradation of LDL by lysosomal cathepsins. In contrast, there was little, if any, inhibition of the neutral protease activity by leupeptin or pepstatin. The acidic protease activity was increased by both DL-dithiothreitol (5 mM) and disodium EDTA (1 mM) whereas the neutral protease activity was increased by dithiothreitol but inhibited partially by EDTA. The possible significance of macrophage neutral and acidic protease activities toward LDL in atherosclerosis needs to be assessed.

Animals↗
PubMed1984

Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids.

Low density lipoprotein (LDL) incubated with cultured endothelial cells from rabbit aorta or human umbilical vein is altered in several ways (EC-modified): (i) It is degraded by macrophages much faster than LDL similarly incubated in the absence of cells or incubated with fibroblasts. (ii) Its electrophoretic mobility is increased. (iii) Its density is increased. We report here that antioxidants completely prevent these changes. We also report that these changes do not take place if transition metals in the medium are chelated with EDTA. During EC-modification as much as 40% of the LDL phosphatidylcholine is degraded to lysophosphatidylcholine by a phospholipase A2-like activity. When incubation conditions in the absence of cells were selected to favor oxidation--for example, by extending the time of incubation of LDL at low concentrations, or by increasing the Cu2+ concentration--LDL underwent changes very similar to those occurring in the presence of cells, including degradation of phosphatidylcholine. Hence, some phospholipase activity appears to be associated with the isolated LDL used in these studies. The results suggest a complex process in which endothelial cells modify LDL by mechanisms involving generation of free radicals and action of phospholipase (s).

Animals↗
PubMed1983

Properties and subcellular localization of elastase-like activities of arterial smooth muscle cells in culture.

The properties and subcellular localization of the elastase-like activities of smooth muscle cells cultured from pig aortas have been investigated. Homogenates of the cells hydrolysed N-succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide, a synthetic substrate for elastases, with a distinct pH optimum of 8.2 and hydrolysed insoluble elastin with a distinct pH optimum of 8.5. Both enzyme activities were directly proportional to the concentration of homogenate in the assay mixture. The activities toward both substrates were inhibited by phenylmethylsulphonyl fluoride and were therefore probably due to a serine peptidase(s). The activities were also inhibited by EDTA and, in a dose-related manner, by alpha 1-antiprotease. Pepstatin, which inhibits cathepsin D, and leupeptin, which inhibits cathepsin B, did not significantly inhibit the elastase-like activities in these cells. The cells were homogenized and a post-nuclear supernatant subjected to sucrose density gradient centrifugation. The distribution of elastase-like activity toward both substrates was similar to that of the plasma membrane marker 5'-nucleotidase, and distinct from those of marker enzymes for the other organelles. Cells were also homogenized with digitonin, which selectively increases the equilibrium density of the plasma membrane. The equilibrium densities of both 5'-nucleotidase and of the elastase-like activities were increased considerably, confirming the plasma membrane localization of the elastase-like activities. The subcellular localization of the elastase-like activities of arterial smooth muscle cells is therefore consistent with a role for them in the degradation of elastin in the normal arterial wall and in atherosclerotic lesions.

Animals↗
PubMed1983

Properties and subcellular localization of adenosine diphosphatase in arterial smooth muscle cells in culture.

The properties and subcellular localization of adenosine diphosphatase (ADPase) activity in smooth muscle cells cultured from pig aortas have been investigated. The pH optimum of ADPase activity was 7.3 and the apparent Km for ADP was 10.3 microM. ADPase activity was inhibited completely by EDTA and was restored by the addition of divalent cations. The enzyme activity was not inhibited by 2-glycerophosphate, a substrate for non-specific phosphatases, nor by levamisole, a specific inhibitor of alkaline phosphatase. Smooth muscle cells were homogenized and a post-nuclear supernatant was applied to a sucrose density gradient in a Beaufay automatic zonal rotor. The distribution of ADPase activity in the density gradient was similar to that of 5'-nucleotidase activity, a marker enzyme for the plasma membrane, and distinct from the distributions of the marker enzymes for the other organelles. When the cells were homogenized in the presence of digitonin, an agent which binds to cholesterol and increases the equilibrium density of the plasma membrane, the modal equilibrium densities of ADPase activity and of 5'-nucleotidase activity were increased to similar extents, thus confirming the plasma membrane localization of ADPase activity.

Animals↗