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D Steinberg

Publications and source records attributed to D Steinberg.

At least 181 records · Page 10Linked to original sources
PubMed1987

Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis.

Previous studies in this laboratory established that low density lipoprotein (LDL) incubated with cultured endothelial cells, smooth muscle cells, or macrophages undergoes free radical-catalyzed oxidative modification that generates lipid peroxides and extensive structural changes in the LDL molecule. The oxidatively modified LDL strongly inhibited chemotactic responses of the mouse resident peritoneal macrophage. The present studies show that this oxidized LDL does not inhibit the motility of mouse monocytes and actually exhibits a chemotactic activity for human monocytes; the chemotactic activity of the oxidized LDL resides in the lipid fraction. These findings allow us to propose a pathogenetic sequence by which elevated plasma LDL levels, followed by oxidative modification in the arterial wall, could sufficiently account for the generation of the lipid-laden foam cells and the initiation of the fatty streak, the earliest well-defined lesion in atherogenesis.

Animals↗
PubMed1987

Lipoproteins and the pathogenesis of atherosclerosis.

It is now clear that hypercholesterolemia can, in some instances, be a necessary and sufficient cause of premature atherosclerosis. This has been best established in patients with familial hypercholesterolemia, a deficiency of the low-density lipoprotein receptor. Although hypercholesterolemia is not the only cause of atherosclerosis, a large body of evidence has identified it as a determining cause in many cases. This article reviews current hypotheses regarding the mechanisms by which hypercholesterolemia accelerates atherogenesis. The role of the foam cell is discussed in detail because it is a characteristic feature of the earliest lesion, the so-called fatty streak. Once thought to derive exclusively from smooth muscle cells, the foam cell is now known to originate in large part from monocytes that enter the artery wall and alter their properties to become tissue macrophages. Recent studies of the biology of the macrophage-derived foam cell are providing new insights into the mechanisms by which it enters the arterial wall and interacts with various classes of native and modified lipoproteins. As our understanding of the biology of the foam cell and its precursors grows, it may become possible to intervene and slow the progress of atherosclerosis by new modalities that might act synergistically with measures to control plasma cholesterol levels.

Arteries↗
PubMed1987

Bronchial reactivity increases soon after the immediate response in dual-responding asthmatic subjects.

To determine whether bronchial reactivity is augmented soon after an allergen-induced immediate asthmatic response, we compared reactivity to histamine before and immediately upon resolution of the immediate asthmatic response in seven subjects with mild asthma who were known to develop dual asthmatic responses after inhalation of Kentucky bluegrass allergen. Using a body plethysmograph and quiet breathing technique, specific airway resistance (SRaw) and reactivity to aerosol histamine were assessed on two days prior to allergenic challenge. The dose of histamine that doubled SRaw (PC200His) was determined by interpolation from histamine dose-response curves. On the day of allergenic challenge, each subject inhaled a concentration that was sufficient to induce a dual asthmatic response. Upon resolution of the immediate asthmatic response (45 to 105 minutes) after allergen, the PC200His in all cases had decreased more than 50 percent of its original value (PC200His for the group was 0.29 +/- 1.42 mg/ml [mean +/- SE], compared to 0.84 +/- 1.23 mg/ml initially). Seven or more days after the allergen, each subject had a PC200His comparable to original values. Our data indicate that airway reactivity in dual-responding asthmatic subjects markedly increases soon after the immediate asthmatic response and much before the late asthmatic response manifests clinically. Whether this early increase in bronchial reactivity is a putative requirement for, or shares common characteristics with the late asthmatic response requires further study.

Adult↗
PubMed1987

Nonenzymatic oxidative cleavage of peptide bonds in apoprotein B-100.

Incubation of low density lipoprotein (LDL) with endothelial cells converts it to a form that is avidly degraded by macrophages via the acetyl LDL receptor. This modification has previously been shown to be accompanied by extensive breakdown of the major LDL protein (apoB-100) to smaller peptides. ApoB-100 is known to undergo partial degradation during isolation and purification which is commonly attributed to proteolytic enzymes derived from plasma or to contaminant bacteria. In the present studies addition of any of ten different inhibitors of proteolytic enzymes failed to inhibit the endothelial cell-induced degradation of LDL apoB-100 or its subsequent enhanced rate of degradation by macrophages (termed biological modification). Conversely, deliberate digestion of LDL with any of five well-characterized proteolytic enzymes degraded apoB-100 extensively but did not cause biological modification. The disappearance of intact apoB-100 during incubation with endothelial cells paralleled the formation of thiobarbituric acid (TBA)-reactive substances and the breakdown could be completely prevented by the addition of antioxidants or metal chelators. Finally, the incubation of LDL with a free radical-generating system (dihydroxyfumaric acid and Fe3+-ADP) in the absence of cells resulted in the breakdown of apoB-100. These results suggest that the breakdown of apoB-100 during oxidative modification of LDL, whether cell-induced or catalyzed by transition metals, is not mediated by proteolytic enzymes but rather is linked to oxidative attack on the polypeptide chain, either directly or secondary to peroxidation of closely associated LDL lipids.

Amino Acids↗
PubMed1987

Turnover and tissue sites of degradation of glucosylated low density lipoprotein in normal and immunized rabbits.

Immunological mechanisms have been implicated in the atherogenic process since immunoglobulins are frequently found in the atherosclerotic aorta. We have previously shown that modifications of homologous low density lipoproteins (LDL) make it immunogenic. In particular we have demonstrated that immunization with homologous nonenzymatically glucosylated LDL (glcLDL) results in the generation of antibodies specific to the derivatized lysine residue, and that such antibodies do not react with native LDL epitopes. In the present study we immunized rabbits with reductively glucosylated rabbit LDL and then determined the effects of the circulating antibodies on the rates of plasma clearance and on the sites of degradation of LDL in which varying degrees of glucosylation had been achieved. In normal chow-fed animals, the plasma clearance of glcLDL was retarded in proportion to the extent of lysine derivatization. In contrast, in immunized animals the clearance of glcLDL was greatly accelerated. When 10% or more of lysine residues were derivatized, clearance of glcLDL was accelerated 50- to 100-fold. Even when only 5% of lysines were derivatized, plasma clearance was accelerated 2- to 3-fold. Cholesterol feeding inhibited LDL clearance from plasma and decreased LDL uptake of LDL receptor-rich tissues. In a similar manner, glucosylation of LDL inhibited its ability to bind to the LDL receptor and redirected sites of LDL degradation away from LDL receptor-rich tissues. Thus degradation of glcLDL by liver and adrenal was markedly diminished. The presence of antibodies to glcLDL also redirected sites of degradation of the modified LDL, primarily to the reticuloendothelial cells of the liver. There was no evidence for specific targeting of glcLDL-immunoglobulin complexes to the aorta; instead they were targeted to the liver. These data suggest that the presence of humoral antibodies to modified LDL acts to rapidly remove such LDL from plasma and specifically targets such complexes to reticuloendothelial cells, primarily in the liver. In this manner such antibodies may serve a useful purpose.

Adrenal Glands↗
PubMed1986

Studies on the mechanism of action of probucol.

Earlier clinical studies suggested that probucol lowers plasma levels of low density lipoprotein (LDL) by increasing the rate of its removal. The drug has been shown to be effective in patients lacking the LDL receptor and, more recently, in LDL receptor-deficient rabbits. These latter studies showed that probucol increased the fractional catabolic rate of LDL by 40% to 50%. This finding in a receptor-deficient model implies that probucol can enhance the removal of LDL cholesterol by alternative pathways. Because the same enhanced removal was seen when LDL from probucol-treated rabbits was injected into untreated rabbits, it appears that the drug somehow alters the metabolic properties of the LDL itself rather than the metabolic behavior of the tissues of treated animals. Whether or not this mechanism explains the action of probucol in man remains to be determined. It has been postulated that the oxidative modification of LDL might contribute to atherogenesis by facilitating lipid accumulation in macrophages (foam cells) and by inhibiting macrophage motility. LDL resists oxidative modification, however, when probucol is added to in vitro incubations or when the LDL itself is isolated from probucol-treated patients. These findings lay the groundwork for evaluating the possible in vivo significance of LDL oxidation.

Animals↗
PubMed1986

Probucol inhibits oxidative modification of low density lipoprotein.

Previous studies have established that low density lipoprotein (LDL) incubated with endothelial cells (EC) undergoes extensive oxidative modification in structure and that the modified LDL is specifically recognized by the acetyl LDL receptor of the macrophage. Thus, in principle, EC-modified LDL could contribute to foam cell formation during atherogenesis. Oxidatively modified LDL is also potentially toxic to EC. The present studies show that addition of probucol during the incubation of LDL with EC prevents the increase in the electrophoretic mobility, the increase in peroxides, and the increase in subsequent susceptibility to macrophage degradation. It has also been shown that oxidation of LDL catalyzed by cupric ion induces many of the same changes occurring during EC modification. Addition of probucol (5 microM) also prevented this copper-catalyzed modification of LDL. Most importantly, samples of LDL isolated from plasma of hypercholesterolemic patients under treatment with conventional dosages of probucol were shown to be highly resistant to oxidative modification either by incubation with endothelial cells or by cupric ion in the absence of cells. The findings suggest the hypothetical but intriguing possibility that probucol, in addition to its recognized effects on plasma LDL levels, may inhibit atherogenesis by limiting oxidative LDL modification and thus foam cell formation and/or EC injury. Other compounds with antioxidant properties might behave similarly.

Animals↗
PubMed1986

Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence.

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.

Animals↗
PubMed1985

Uptake of high-density lipoprotein-associated apoprotein A-I and cholesterol esters by 16 tissues of the rat in vivo and by adrenal cells and hepatocytes in vitro.

The uptake of high-density lipoprotein (HDL)-associated apolipoprotein A-I and cholesterol esters was estimated in 16 tissues of the rat using rat HDL doubly labeled with nondegradable tracers; covalently attached 125I-tyramine-cellobiose traced apo-A-I, and [3H]cholesteryl linoleyl ether traced cholesterol esters. Both labels remained associated with the HDL fraction in the plasma, adequately traced their unlabeled counterparts, and were well trapped at their sites of uptake. Cholesteryl ether was taken up at a greater fractional rate than apo-A-I by adrenal, ovary, and liver: 7-fold, 4-fold, and 2-fold greater, respectively. The rates of uptake of cholesteryl ether and apo-A-I were about equal in the other tissues (except kidney). The disproportionate uptake of HDL cholesteryl ether relative to HDL apo-A-I was also observed in primary cultures of rat adrenal cells and hepatocytes. Uptake of both moieties in both cell types showed saturability. Both the absolute rate of uptake of [3H]cholesteryl ether and the ratio of ether uptake to apo-A-I uptake were greater in adrenal cells than in hepatocytes, consonant with the in vivo observations. Very similar results were obtained using HDL biologically labeled with [3H]cholesterol esters. The disproportionate uptake of [3H]cholesteryl ether was not significantly decreased by depletion of apo-E from the HDL nor by reductive methylation of the apo-E to block its recognition by receptors. However, apo-A-I uptake was decreased, suggesting that apo-E mediates the uptake of particles containing apo-A-I but does not contribute to the disproportionate uptake of [3H]cholesteryl ether.

Adrenal Glands↗
PubMed1985

Endothelial cell-derived chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein.

Cultured rabbit and bovine aortic endothelial cells generated chemotactic activity for mouse resident peritoneal macrophages, demonstrable in the conditioned medium. This chemotactic activity was heat stable and was not extracted into chloroform/methanol. It was inhibited by addition of endothelial cell-modified low density lipoprotein (EC-modified LDL), a form of LDL shown previously to contain peroxidized lipids, increased lysophosphatidylcholine, and partially degraded apoprotein B. The chemotactic activity was also inhibited by LDL previously oxidized in the absence of cells with 5 microM Cu2+. Inhibitory activity was present in the lipid extract of EC-modified LDL but not in that of native LDL, presumably representing peroxidized lipid components. EC-modified LDL also inhibited the chemotactic activity of zymosan-activated serum. Because EC-modified LDL is taken up in part by way of the acetyl-LDL receptor, the effects of acetyl-LDL were tested. Rather than inhibiting chemotaxis, acetyl LDL showed intrinsic positive chemotactic activity as did also fucoidin and polyinosinic acid, both of which also interact with the acetyl-LDL receptor. These studies suggest mechanisms by which macrophages may be recruited into the subendothelial space by endothelial cell-derived chemotactic factors or by natural polyanions structurally related to fucoidin or polyinosinic acid and then become "trapped" there because of the inhibitory effects of peroxidized lipid components in modified forms of LDL.

Animals↗
PubMed1985

Essential role of phospholipase A2 activity in endothelial cell-induced modification of low density lipoprotein.

Previous studies have established that incubation of low density lipoprotein (LDL) with cultured endothelial cells (EC) converts it to a new form (EC-modified LDL) that is now recognized by a specific receptor on macrophages (the acetyl LDL receptor) and is taken up and degraded 3-10 times more rapidly than native LDL (biological modification). The formation of EC-modified LDL depended on generation of free radicals with consequent peroxidation of LDL lipids and was accompanied by extensive hydrolysis of LDL phosphatidylcholine at the 2-position. The present studies show that p-bromophenacyl bromide, a site-specific irreversible inhibitor of phospholipase A2 activity, blocks this hydrolysis and, at the same time, the enhanced macrophage degradation. We show further that during EC modification the apoprotein B of LDL undergoes considerable modification and that this also is prevented by the phospholipase inhibitor. Finally, as reported previously, changes similar to those observed on incubation of LDL with EC can be induced by incubation in the absence of cells but in the presence of a sufficiently high concentration of Cu2+. This also is accompanied by hydrolysis of phosphatidylcholine at the 2-position and breakdown of apoprotein B. These changes are also inhibited by p-bromophenacyl bromide, suggesting the presence of a phospholipase A2 activity associated with LDL as it is isolated. A hypothesis is presented linking lipid peroxidation, phosphatidylcholine hydrolysis, and changes in the LDL apoprotein during EC modification.

Acetophenones↗
PubMed1984

Sites and mechanisms of uptake and degradation of high density and low density lipoproteins.

In spite of much progress in understanding the mechanisms of lipoprotein metabolism at the cellular level, much less is known quantitatively about what actually occurs in vivo. A new tool for understanding the catabolic fates of lipoproteins in vivo is the use of noncatabolizable ligands for labeling proteins. These ligands are incorporated into cells with the labeled protein. The protein is catabolized but the ligand remains intact and trapped at the sites of hydrolysis as a cumulative marker of the extent of protein uptake. This tool was first applied to LDL in pigs, rats, and rabbits. In all three, hepatic catabolism predominated. Adrenal gland was the most active per unit wet weight. The trapped label methodology was also applied to the apoA-I component of HDL in rats. [3H]Cholesteryl ethers were also incorporated as noncatabolized, intracellularly trapped tracers of cholesteryl esters. Liver was the predominant site of HDL cholesteryl ether uptake, consistent with a role of HDL in reverse cholesterol transport; adrenal was the most active per unit wet weight. In adrenal, ovary, and liver, the cholesteryl ether tracer was taken up at a much greater rate than apoA-I, suggesting a mechanism for cholesteryl ester uptake independent of holo-particle uptake. Such a mechanism was verified in primary cultures of rat adrenal cells and hepatocytes.

Animals↗
PubMed1984

Lipoprotein synthesis and secretion by cultured rat hepatocytes. Parallel inhibition of secretion of VLDL, HDL and albumin by monensin.

The biosynthesis and secretion of very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) by cultured normal rat hepatocytes was investigated with particular emphasis on its modification by monensin. This acidic ionophore coordinately inhibited the rates of secretion of the several VLDL apolipoproteins and the VLDL lipids, suggesting an effect late in the process of biosynthesis and secretion, probably at the stage of exiting from the Golgi apparatus. The secretion of immunoreactive albumin into the medium was comparably inhibited, implying that the pathway and mechanisms involved in albumin secretion may be closely similar to those for VLDL synthesis and secretion. Secretion of phospholipids and of apolipoproteins E and A-I in the HDL fraction increased progressively with time over 18 h in control incubations but was strongly inhibited by monensin. During extended incubation with monensin at high concentrations (10 microM), there was a net release to the medium of a number of hepatocyte proteins, including some that comigrated with apolipoprotein A-I and apolipoprotein C, making it appear that monensin increased the secretion of these apolipoproteins. However, using labeled amino acids, it was shown by autoradiography and by immunoprecipitation that secretion of newly-synthesized, radioactive apolipoprotein A-I and apolipoprotein C was actually inhibited by monensin. These results are compatible with the conclusion that HDL synthesis and secretion may occur by mechanisms closely related to those for synthesis and secretion of albumin and VLDL.

Amino Acids↗