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Biomedical subjects

A Chait

Publications and source records attributed to A Chait.

At least 91 records · Page 5Linked to original sources
PubMed1993

Oxidation of low density lipoprotein by thiols: superoxide-dependent and -independent mechanisms.

Oxidatively damaged low density lipoprotein (LDL) may cause macrophages to accumulate cholesterol in an unregulated manner, initiating the development of atherosclerotic lesions. Cultured smooth muscle cells oxidize LDL by a superoxide (O2.-)-dependent mechanism that requires L-cystine and redox-active transition metal ions in the incubation medium. To test the hypothesis that cellular reduction of L-cystine to a thiol might be involved, we exposed LDL to L-cysteine, glutathione, and D,L-homocysteine. In a cell-free system each thiol modified LDL by a pathway that required either Cu2+ or Fe3+. Thiol- and Cu(2+)-modified LDL underwent lipid peroxidation and exhibited a number of properties of cell-modified LDL, including increased mobility on agarose gel electrophoresis and fragmentation of apolipoprotein B-100. Superoxide dismutase inhibited modification of LDL by L-cysteine/Cu2+, whereas catalase and mannitol were without effect. In striking contrast, superoxide dismutase had little effect on oxidation of LDL by Cu2+ and either homocysteine or glutathione. Moreover, only L-cysteine/Cu(2+)-modified 125I-labeled LDL was degraded more rapidly than 125I-labeled LDL by human monocyte-derived macrophages: superoxide dismutase in the reaction mixture blocked the facilitated uptake of L-cysteine/Cu(2+)-modified 125I-labeled LDL, suggesting involvement of O2.-. These results indicate that LDL oxidation by L-cysteine and Cu2+ requires O2.- but not H2O2 or hydroxyl radical. The reaction may involve the metal ion-dependent formation of L-cystine radical anion which is oxidized by oxygen, yielding O2.- and the disulfide. LDL modified by L-cysteine and smooth muscle cells exhibit similar physical and biological properties, indicating that thiol-dependent generation of O2.- may be the oxidative mechanism in both systems. Thiols also promote lipid peroxidation by O2(.-)-independent reactions but human macrophages fail to rapidly degrade these oxidized LDLs.

Cell-Free System↗
PubMed1992

Lipoprotein lipase is synthesized by macrophage-derived foam cells in human coronary atherosclerotic plaques.

Lipoprotein lipase (LPL), hydrolyzes the core triglycerides of lipoproteins, thereby playing a role in their maturation. LPL may be important in the metabolic pathways that lead to atherosclerosis, since it is secreted in vitro by both of the predominant cell types of the atherosclerotic plaque, i.e., macrophages and smooth muscle cells. Because of uncertainty concerning the primary cellular source of LPL in atherosclerotic lesions, in situ hybridization assays for LPL mRNA were performed on 12 coronary arteries obtained from six cardiac allograft recipients. Macrophages and smooth muscle cells were identified on adjacent sections with cell-specific antibodies and foam cells were identified morphologically. LPL protein was localized using a polyclonal antibody. LPL mRNA was produced by a proportion of plaque macrophages, particularly macrophage-derived foam cells, but was not detected in association with any intimal or medial smooth muscle cells. These findings were confirmed by combined immunocytochemistry and in situ hybridization on the same tissue sections. LPL protein was detected in association with macrophage-derived foam cells, endothelial cells, adventitial adipocytes, and medial smooth muscle cells, and, to a lesser extent, in intimal smooth muscle cells and media underlying well-developed plaque. These results indicate that macrophage-derived foam cells are the primary source of LPL in atherosclerotic plaques and are consistent with a role for LPL in the pathogenesis of atherosclerosis.

Arteriosclerosis↗
PubMed1992

Immunohistochemical distribution of lipoprotein epitopes in xanthomata from patients with familial hypercholesterolemia.

Human xanthomas derived from four subjects with familial hypercholesterolemia (3 homozygotes and 1 heterozygote) were studied by immunohistochemical methods to determine the presence and distribution of lipoproteins, which have been implicated in the pathogenesis of atherosclerosis. Oxidatively modified low-density lipoprotein (OxLDL) epitopes detected with anti-OxLDL monoclonal antibodies, appeared to have a similar distribution in xanthomata to that of macrophages, detected by a cell-specific monoclonal antibody. Double antibody labeling with both an anti-macrophage antibody and an anti-OxLDL antibody demonstrated that OxLDL epitopes are associated with macrophages and occurred intracellularly. Low-density lipoprotein (LDL) epitopes were detected extracellularly, with a distribution that was different from that of OxLDL. In addition, apo(a) epitopes detected by an apo(a) specific monoclonal antibody, had a distribution similar to that of LDL in the dermis and subcutaneous tissues. The observed epitope distribution of LDL, OxLDL, or apo(a) was the same regardless of the method of treatment of the patients from whom the xanthomas were obtained (probucol, simvastatin, LDL apheresis). These findings suggest that OxLDL is likely to play a pathogenetic role in the lipid accumulation by macrophages in xanthomas, and suggest that Lp(a) also may play a role in their pathogenesis.

Adolescent↗
PubMed1992

Chylomicronemia syndrome.

This chapter has outlined the reasons for the development of marked elevations of triglycerides that can be associated with the chylomicronemia syndrome. The clinical features of the syndrome have been discussed, with particular emphasis on chylomicron-induced pancreatitis, since this can be life-threatening. An approach to the diagnosis and management of this syndrome has been provided, with emphasis on the need for long-term follow-up and compliance to keep plasma triglycerides to a reasonable level and thereby avoid recurrent clinical complications due to chronic chylomicronemia.

Acute Disease↗
PubMed1991

Phagocytosis of opsonized oil droplets by neutrophils. Adaptation to a microtiter plate format.

A previously described assay for neutrophil phagocytosis of oil droplets labeled with the lipophilic red dye, oil red O, has been adapted to a microtiter plate format. Oil laden neutrophils are fixed to the plate with glutaraldehyde, washed free of uningested oil, and evaluated for degree of oil red O uptake by direct determination of light absorption at 540 nm in a microtiter plate reader. Advantages of the modified procedure include the ability to determine rates of phagocytosis (multiple determinations) under diverse conditions, simultaneously and with greater facility. Multiple centrifugation steps are eliminated and the requirement for extraction of oil red O from each cell pellet prior to quantitation becomes optional. The system has been evaluated for oil droplets emulsified with lipopolysaccharide alone or with lipopolysaccharide together with bovine serum albumin. For droplets emulsified with lipopolysaccharide alone, efficient opsonization was accomplished with fresh serum alone, through heat labile, presumably complement-dependent, pathways. For droplets emulsified with lipopolysaccharide and bovine serum albumin, specific antiserum to bovine serum albumin was a considerably more efficient opsonin than non-immune fresh serum. A third system is described in which tetanus toxoid was introduced into oil droplets that had been previously emulsified with lipopolysaccharide. Inclusion of toxoid antigens rendered the oil droplets susceptible to opsonization with tetanus immune globulin, making the assay system possibly applicable to evaluation of immunoglobulin function in humans.

Azo Compounds↗
PubMed1991

The influence of oxidatively modified low density lipoproteins on expression of platelet-derived growth factor by human monocyte-derived macrophages.

Platelet-derived growth factor (PDGF) is secreted by several cells that participate in the process of atherogenesis, including arterial wall monocyte-derived macrophages. Macrophages in human and non-human primate lesions have recently been demonstrated to contain PDGF-B chain protein in situ. In developing lesions of atherosclerosis, macrophages take up and metabolize modified lipoproteins, leading to lipid accumulation and foam cell formation. Oxidatively modified low density lipoproteins (LDL) have been implicated in atherogenesis and have been demonstrated in atherosclerotic lesions. The effects of the uptake of various forms of modified LDL on PDGF gene expression, synthesis, and secretion in adherent cultures of human blood monocyte-derived macrophages were examined. LDL oxidized in a cell-free system in the presence of air and copper inhibited the constitutive expression of PDGF-B mRNA and secretion of PDGF in a dose-dependent fashion. Oxidatively modified LDL also attenuated lipopolysaccharide-induced PDGF-B mRNA expression. These changes were unrelated to the mechanism of lipid uptake and the degree of lipid loading and were detectable within 2 h of exposure to oxidized LDL. The degree of inhibition of both basal and lipopolysaccharide-induced PDGF-B-chain expression increased with the extent of LDL oxidation. Monocyte-derived macrophages exposed to acetylated LDL or LDL aggregates accumulated more cholesterol than cells treated with oxidized LDL, but PDGF expression was not consistently altered. Thus, uptake of a product or products of LDL oxidation modulates the expression and secretion of one of the principal macrophage-derived growth factors, PDGF. This modulation may influence chemotaxis and mitogenesis of smooth muscle cells locally in the artery wall during atherogenesis.

Antioxidants↗
PubMed1991

Transcriptional activation of the lipoprotein lipase gene in macrophages by dexamethasone.

The effect of dexamethasone on lipoprotein lipase (LPL) gene expression during macrophage differentiation was investigated by using the human monocytic leukemia cell line THP-1 and human monocyte-derived macrophages. Addition of dexamethasone to THP-1 cells increased steady-state levels of LPL mRNA and LPL mass accumulation in the medium during PMA-induced differentiation by 4-fold. Studies with human monocyte-derived macrophages showed a similar effect of dexamethasone on LPL expression. Peak LPL mRNA levels were achieved 24-h post-dexamethasone addition to THP-1 cells. Optimal stimulation of LPL mRNA occurred when dexamethasone was added 24 h after induction with PMA. Thereafter, there was rapid decline in responsiveness to dexamethasone. Induction of LPL mRNA in THP-1 cells was completely blocked by actinomycin D, suggesting that induction was transcription dependent. The stability of LPL mRNA was not influenced by dexamethasone. Treatment of THP-1 cells with PMA led to a 2-fold increase in specific binding of dexamethasone and a 4-fold increase in glucocorticoid receptor mRNA within 12 h. Thus, dexamethasone stimulates LPL gene expression during differentiation of human macrophages, a process that involves induction of glucocorticoid receptor synthesis and activation.

Blotting, Northern↗
PubMed1990

Anatomical mapping of brain sites involved in the antinociceptive effects of ketoprofen.

Ketoprofen, a non-steroidal anti-inflammatory drug, has analgesic effects in animals and humans through a peripheral as well as a central action. This study was designed to determine which brain sites are involved in the central analgesic action of ketoprofen, by using the hot-plate test. Latencies to the first hindpaw lick were recorded in animals receiving local cerebral injections of ketoprofen (10 micrograms in 0.3 microliters) or a control solution (saline). Nineteen brain sites were tested. A significant analgesia was obtained in the 8 following sites: central gray, centro-medial nucleus of the thalamus, nucleus reuniens, dorso-lateral geniculate nucleus, medial geniculate nucleus, dorso-medial and ventro-medial nuclei of the hypothalamus, posterior hypothalamic nucleus and lateral vestibular nucleus. A slight analgesia, which did not reach significance, was observed in three structures: dorsal raphé, raphé magnus and ventral postero-medial thalamic nucleus. No analgesia was observed in other sites: centro-lateral nucleus of the thalamus, posterior thalamic nuclear group, parafascicular nucleus, bed nucleus of the stria terminalis, mesencephalic tegmentum, nucleus of the tractus solitarius, spinal trigeminal complex, and brainstem reticular formation. Therefore ketoprofen seems to be centrally active mostly at the level of several integrative and non-specific structures.

Animals↗
PubMed1990

Regulation of LTP-I secretion from human monocyte-derived macrophages by differentiation and cholesterol accumulation in vitro.

Human macrophages in vitro synthesize and secrete the cholesteryl ester (CE) transfer protein, LTP-I. The effect of differentiation of monocyte-to-macrophage on the synthesis and secretion of LTP-I cholesteryl ester transfer activity was investigated. One marker of macrophage differentiation is expression of the 'scavenger' receptor, which mediates macrophage uptake and degradation of acetylated low-density lipoprotein. Monocytes secreted very little detectable CE transfer activity in the first 24 h following cell isolation. Both CE transfer activity and scavenger receptor activity increased with time in culture. Thus, although circulating monocytes probably do not secrete CE transfer activity, tissue macrophages such as hepatic Kupffer cells may contribute to plasma CE transfer activity. Resident macrophages of the arterial wall are derived from circulating monocytes which enter the vessel wall where they differentiate into macrophages. Such macrophages are the principal source of lipid-laden foam cells of the atherosclerotic plaque. Cholesterol accumulation results when uptake of lipoprotein cholesterol overwhelms the capacity of macrophages to excrete cholesterol. Since LTP-I is postulated to function in reverse cholesterol transport, the effect on LTP-I secretion of loading macrophages with cholesterol was determined after exposure of macrophages to acetylated-LDL or free cholesterol (FC). Cholesterol loading by both these maneuvers resulted in dose-dependent increases in macrophage secretion of CE transfer activity, and there was a significant positive correlation between CE transfer activity secreted and accumulation of CE. Thus, LTP-I may function at the cellular level in maintenance of lipid homeostasis: macrophage LTP-I secretion may be a protective mechanism in response to excess cholesterol accumulation in resident macrophages of the arterial wall.

Blotting, Western↗
PubMed1990

Acquired hyperlipidemia (secondary dyslipoproteinemias).

Acquired hyperlipidemia (secondary dyslipoproteinemias) results from underlying disorders that lead to alterations in plasma lipid and lipoprotein metabolism. Secondary dyslipoproteinemias may mimic primary forms of hyperlipidemia and can have similar consequences. They may result in increased predisposition to premature atherosclerosis or, when associated with marked hypertriglyceridemia, may lead to the development of pancreatitis and other features of the chylomicronemia syndrome. Diabetes mellitus and use of drugs such as diuretics, beta blockers, and estrogens are commonly encountered causes of secondary dyslipoproteinemia. Other conditions leading to acquired hyperlipidemia include hypothyroidism, renal failure, nephrotic syndrome, alcohol usage, and some rare endocrine and metabolic disorders. When secondary and familial forms of hypertriglyceridemia coexist, triglyceride removal mechanisms may be saturated and marked hypertriglyceridemia with fasting chylomicronemia might ensue. Treatment of the underlying condition, when possible, or discontinuation of the offending drugs usually leads to an improvement in the hyperlipidemia. Specific lipid-lowering therapy may be required in certain circumstances.

Diabetes Complications↗
PubMed1989

Lipoprotein lipase gene expression in THP-1 cells.

Lipoprotein lipase (LPL) mRNA levels are under the control of signals that activate phospholipase C, resulting in activation of protein kinase C (PKC) and mobilization of intracellular Ca2+ in the human monocytic leukemia cell line THP-1. Induction of LPL in THP-1 cells appears to be mediated by PKC since it was affected by both phorbol 12-myristate 13-acetate (PMA) and a diacylglycerol analogue. This induction was blocked by the specific PKC inhibitor H-7. Although Ca2+ mobilization by the ionophore A23187 also induced LPL mRNA, the mechanism is most likely independent of activation of the Ca2+/calmodulin protein kinase. Depletion of cells of PKC made them refractory to induction by A23187, suggesting that Ca2+ mobilization acts by activating PKC. Addition of cycloheximide (CHX) to undifferentiated THP-1 cells resulted in a transient increase in steady-state mRNA levels (3-fold). Sustained superinduction of LPL mRNA occurred when PMA and CHX were added simultaneously. These results suggest that the level of LPL mRNA is regulated either by a labile regulatory protein, which represses transcription of the LPL gene, or by a protein affecting mRNA stability.

Calcium↗
PubMed1989

Regulation of the low density lipoprotein receptor and hydroxymethylglutaryl coenzyme A reductase genes by protein kinase C and a putative negative regulatory protein.

Transcription of the low density lipoprotein receptor (LDL-R) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase genes was rapidly and transiently induced (8.5- and 2.3-fold, respectively) early during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of the human monocytic leukemia cell line THP-1. The levels of mRNA coding for LDL-R and HMG-CoA reductase increased soon after induction, reached a maximum (12- and 7-fold increase, respectively) in 2-3 hr, and then rapidly returned to the low constitutive levels observed before induction. The stability of LDL-R mRNA did not change significantly during differentiation, whereas that of HMG-CoA reductase mRNA decreased by about 5-fold 6 hr after the addition of PMA. Transcriptional induction of both LDL-R and HMG-CoA reductase genes (5.6- and 2-fold, respectively) was also observed when undifferentiated cells were treated with cycloheximide (CHX), resulting in a transient increase in steady-state mRNA (7- and 3-fold, respectively). These results suggest that expression of the two genes is maintained at low constitutive levels in uninduced THP-1 cells by a protein with a short half-life. Superinduction of both genes occurred when PMA and CHX were added simultaneously. The induction of LDL-R and HMG-CoA reductase mRNAs during early macrophage differentiation is mediated by protein kinase C. It is hypothesized that protein kinase C acts directly or indirectly to inactivate the labile negative regulatory protein. Induction of LDL-R mRNA was also observed when the human hepatocarcinoma cell line Hep G2 was treated with PMA and CHX, suggesting that this mechanism of regulation may exist in several cell types.

Cell Line↗
PubMed1989

Phagocytosis of aggregated lipoprotein by macrophages: low density lipoprotein receptor-dependent foam-cell formation.

Low density lipoprotein (LDL) modified by incubation with phospholipase C (PLC-LDL) aggregates in solution and is rapidly taken up and degraded by human and mouse macrophages, producing foam cells in vitro. Human, mouse, and rabbit macrophages degraded 125I-labeled PLC-LDL (125I-PLC-LDL) more rapidly than native 125I-labeled LDL (125I-LDL), while nonphagocytic cells such as human fibroblasts and bovine aortic endothelial cells degraded 125I-PLC-LDL more slowly than 125I-LDL. This suggested the mechanism for internalization of PLC-LDL was phagocytosis. When examined by electron microscopy, mouse peritoneal macrophages appeared to be phagocytosing PLC-LDL. The uptake and degradation of 125I-PLC-LDL by human macrophages was inhibited greater than 80% by the monoclonal antibody C7 (IgG2b) produced by hybridoma C7, which blocks the ligand binding domain of the LDL receptor. Similarly, methylation of 125I-LDL (125I-MeLDL) prior to treatment with phospholipase C decreased its subsequent uptake and degradation by human macrophages by greater than 90%. The uptake and degradation of phospholipase C-modified 125I-MeLDL by macrophages could be restored by incubation of the methylated lipoprotein with apoprotein E, a ligand recognized by the LDL receptor. These results indicate that macrophages internalize PLC-LDL by LDL receptor-dependent phagocytosis.

Animals↗
PubMed1989

Involvement of second messengers in regulation of the low-density lipoprotein receptor gene.

Transcription of the low-density lipoprotein receptor (LDL-R) gene in the human monocytic leukemic cell line THP-1 and in the human hepatocarcinoma cell line Hep-G2 is regulated by second messengers of the diacylglycerol-protein kinase C (DAG-PKC), inositol 1,4,5-triphosphate-Ca2+, and cyclic AMP pathways. Exogenous phospholipase C (which releases DAG and inositol 1,4,5-triphosphate), PKC activators (phorbol esters and DAG), Ca2+ ionophores, and a cyclic AMP analog all transiently induced accumulation of LDL-R mRNA. The effects of these three signal-transducing pathways were to a large extent additive. Furthermore, PKC stimulation effected an increase in LDL binding, which suggested that the increase in LDL-R mRNA resulted in an increase in functional cell surface receptor activity. These results suggest that uptake of cholesterol by these cells is under control of both intracellular cholesterol levels and external signals.

Calcimycin↗
PubMed1989

Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein.

The rate of degradation of oxidatively modified low density lipoprotein (Ox-LDL) by human endothelial cells was similar to that of unmodified low density lipoprotein (LDL), and was approximately 2-fold greater than the rate of degradation of acetylated LDL (Ac-LDL). While LDL and Ac-LDL both stimulated cholesterol esterification in endothelial cells, Ox-LDL inhibited cholesterol esterification by 34%, demonstrating a dissociation between the degradation of Ox-LDL and its ability to stimulate cholesterol esterification. Further, while LDL and Ac-LDL resulted in a 5- and 15-fold increase in cholesteryl ester accumulation, respectively, Ox-LDL caused only a 1.3-fold increase in cholesteryl ester mass. These differences could be accounted for, in part, by the reduced cholesteryl ester content of Ox-LDL. However, when endothelial cells were incubated with Ac-LDL in the presence and absence of Ox-LDL, Ox-LDL led to a dose-dependent inhibition of cholesterol esterification without affecting the degradation of Ac-LDL. This inhibitory effect of Ox-LDL on cholesteryl ester synthesis was also manifest in normal human skin fibroblasts incubated with LDL and in LDL-receptor-negative fibroblasts incubated with unesterified cholesterol to stimulate cholesterol esterification. Further, the lipid extract from Ox-LDL inhibited cholesterol esterification in LDL-receptor negative fibroblasts. These findings suggest that the inhibition of cholesterol esterification by oxidized LDL is independent of the LDL and scavenger receptors and may be a result of translocation of a lipid component of oxidatively modified LDL across the cell membrane.

Acetylation↗