Biomedical subjects

N Kondratenko

Publications and source records attributed to N Kondratenko.

8 recordsLinked to original sources

PubMed1998

Identification of the lectin-like receptor for oxidized low-density lipoprotein in human macrophages and its potential role as a scavenger receptor.

A new receptor for oxidized low-density lipoprotein (LDL), lectin-like oxidized LDL receptor-1 (LOX-1), has recently been cloned from bovine endothelial cells and human lung. A limited tissue-distribution study suggested that the protein was mainly produced by the vascular endothelium. In the present study we demonstrate that LOX-1 is also expressed in macrophages, where it may function as a scavenger receptor. LOX-1 was not detected in undifferentiated THP-1 cells or in freshly isolated human blood monocytes. However, mature human monocyte-derived macrophages and differentiated THP-1 cells showed high levels of LOX-1 transcripts. Consistent with these results, immunofluorescence staining and FACS analysis demonstrated that LOX-1 protein is expressed on the plasma membrane of macrophages. Western-blot analysis of membranes from macrophages (but not those from monocytes) identified a single band, with an apparent molecular mass of about 40 kDa, that displayed oxidized LDL-binding activity. These results suggest that differentiation induces the expression of LOX-1 in macrophages, where it may play a role as a scavenger receptor and/or a receptor for oxidized LDL.

Animals↗

PubMed1998

Minimally oxidized low-density lipoprotein increases expression of scavenger receptor A, CD36, and macrosialin in resident mouse peritoneal macrophages.

Fully oxidized LDL (OxLDL) is believed to contribute to atherogenesis in part by virtue of uptake into macrophages via specific scavenger receptors. This phenomenon results in the formation of cholesterol-loaded foam cells, a major component of atherosclerotic lesions. The present study is directed at examining the effects of OxLDL and minimally oxidized LDL (MM-LDL) on scavenger receptor expression and activity in mouse peritoneal resident macrophages. Macrophages were preincubated with MM-LDL or OxLDL at concentrations of 25 or 50 microg/mL for 24 to 48 hours, after which their ability to bind and take up 125I-OxLDL or 125I-acetylated LDL (AcLDL) was determined. MM-LDL pretreatment induced a clear increase of cell association and degradation of 125I-OxLDL and 125I-AcLDL. Pretreatment with OxLDL also enhanced scavenger receptor activity, but to a lesser degree. Neither native LDL nor AcLDL had any effect. Scatchard analysis showed that preincubation with 50 microg/mL MM-LDL for 48 hours increased the Bmax of 125I-OxLDL and 125I-AcLDL by 139% and 154%, respectively, without significantly changing their affinity. Lipids extracted from MM-LDL also significantly induced scavenger receptor activity, but to a lesser extent than did intact MM-LDL. MM-LDL pretreatment increased both mRNA levels and protein levels of scavenger receptor A, CD36, and macrosialin. On the other hand, OxLDL pretreatment increased expression of macrosialin only. These results, showing that MM-LDL can upregulate scavenger receptor expression in mouse resident peritoneal macrophages, suggest that clearance of OxLDL by macrophages in lesions is more effective, in part because the OxLDL precursor, MM-LDL, primes the macrophage for foam cell generation.

Animals↗

PubMed1997

Macrophages lacking scavenger receptor A show a decrease in binding and uptake of acetylated low-density lipoprotein and of apoptotic thymocytes, but not of oxidatively damaged red blood cells.

Macrophage binding of oxidatively damaged red blood cells (OxRBC) and apoptotic thymocytes correlates in many instances with a loss of phospholipid bilayer asymmetry, i.e., with an increase in expression of phosphatidylserine on the outer leaflet of the plasma membrane. Oxidatively modified LDL (OxLDL) can compete for the binding of these ligands to macrophages. However, the receptor(s) responsible remains to be identified. The present studies show that mouse peritoneal macrophages totally lacking scavenger receptor A (SRA) bound OxRBC just as effectively as wild-type macrophages, whereas their binding and uptake of acetyl LDL was reduced by more than 80%. Binding of apoptotic thymocytes and binding of OxLDL were also reduced, but only by 20-30%. We conclude that SRA is not involved in the recognition of phosphatidylserine-rich membranes but contributes to the binding of OxLDL and apoptotic thymocytes. The binding of OxRBC was almost totally calcium-dependent, whereas the binding of apoptotic thymocytes was not, suggesting that the mechanisms involved in their uptake by macrophages under these conditions were different.

Animals↗

PubMed1997

Characterization of CLA-1, a human homologue of rodent scavenger receptor BI, as a receptor for high density lipoprotein and apoptotic thymocytes.

Recently, a murine scavenger receptor type B class I (SR-BI) was identified that binds high density lipoprotein (HDL) and mediates the selective uptake of cholesterol esters. The human CD36 and LIMPII analogous-1 (CLA-1) receptor shows high sequence homology with SR-BI, but their functional relationship has not been determined. Transfected cells expressing CLA-1 bound HDL with a Kd of about 35 microg/ml, similar to the Kd for HDL binding to rodent SR-BI. This binding resulted in an intracellular accumulation of HDL-derived [3H]cholesterol esters without internalization or degradation of 125I-apolipoprotein. CLA-1 was strongly expressed in the adrenal gland and was also abundant in liver and testis, suggesting that CLA-1, like SR-BI, could play a role in the metabolism of HDL. However, CLA-1 was also expressed in monocytes and, like SR-BI, recognized modified forms of low density lipoproteins as well as native LDL and anionic phospholipids. These findings suggest that CLA-1 might have additional physiological functions. We found that CLA-1 recognizes apoptotic thymocytes. Our results demonstrate that CLA-1, a close structural homologue of SR-BI, is also functionally related to SR-BI and may play an important role as a "docking receptor" for HDL in connection with selective uptake of cholesterol esters. An additional role in recognition of damaged cells is suggested by these studies.

Animals↗

PubMed1996

Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein.

We have previously identified a 94- to 97-kDa oxidized low density lipoprotein (LDL)-binding protein in mouse macrophages as macrosialin (MS), a member of the lamp family. Earlier immunostaining studies have shown that MS and its human homolog, CD68, are predominantly intracellular proteins. However, using sensitive techniques such as flow cytometry (FACS) and cell-surface-specific biotinylation, we now show that there is significant surface expression of these proteins. FACS analysis of intact cells using mAb FA/11 showed small but definite surface expression of MS in resident mouse peritoneal macrophages but this was greatly enhanced with thioglycollate elicitation. Biotinylation of intact cells and detergent-solubilized cell preparations followed by immunoprecipitation revealed 10-15% of the total MS content of elicited macrophages on the plasma membrane. Similar results were obtained with untreated RAW 264.7 cells. FACS analysis of intact THP-1 monocytic cells showed minimal surface expression of CD68 on unactivated cells (4% of total cell content). Stimulation with phorbol 12-myristate 13-acetate increased both surface and total CD68 expression considerably. Furthermore, the specific binding at 4 degrees C and uptake at 37 degrees C of 125I-labeled oxidized LDL by activated THP-1 cells was inhibited by 30-50% by CD68 mAbs KP-1 and EBM-11. Thus, although the surface expression of MS/CD68 at steady-state represents only a small percentage of their total cellular content, these proteins can play a significant role in oxidized LDL uptake by activated macrophages in vitro and could contribute to foam cell formation in atherosclerotic lesions.

Animals↗

PubMed1995

A macrophage receptor for oxidized low density lipoprotein distinct from the receptor for acetyl low density lipoprotein: partial purification and role in recognition of oxidatively damaged cells.

The binding and uptake of oxidatively modified low density lipoprotein (OxLDL) by mouse peritoneal macrophages occurs, in part, via the well characterized acetyl LDL receptor. However, several lines of evidence indicate that as much as 30-70% of the uptake can occur via a distinct receptor that recognizes OxLDL with a higher affinity than it recognizes acetyl LDL. We describe the partial purification and characterization of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that specifically binds OxLDL. This receptor is shown to be distinct from the acetyl LDL receptor as well as from two other macrophage proteins that also bind OxLDL--the Fc gamma RII receptor and CD36. We suggest that this OxLDL-binding membrane protein participates in uptake of OxLDL by murine macrophages and also represents a receptor responsible for macrophage binding and phagocytosis of oxidatively damaged cells.

Animals↗

PubMed1992

Direct modification of low density lipoprotein by the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid.

We have previously reported that the spin trap alpha-phenyl-tert-butyl nitrone (PBN) inhibited the oxidative modification of low density lipoprotein (LDL) (Kalyanaraman, B., Antholine, W.E. and Parthasarathy, S. (1990) Biochim. Biophys. Acta 1035, 286-292). In the present study, we report that 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS), a water-soluble spin trap, also inhibited the oxidation of LDL as measured by the formation of thiobarbituric acid reactive substances (TBARS). However, when compared with LDL incubated without DBNBS, the DBNBS-incubated LDL showed increased negative charge on agarose gel electrophoresis and was avidly degraded by mouse peritoneal macrophages. Despite the suggestion of biological modification, there was no decrease in lysine-amino groups in DBNBS-incubated LDL. Furthermore, reductively methylated LDL in which more than 85% of the amino group of lysines was blocked, was also modified by DBNBS. A sulfonic acid analog of PBN failed to modify LDL in a similar manner, suggesting that the presence of sulfonic acid alone does not ensure modification. When LDL was incubated with DBNBS, radical adducts associated with both lipid and protein were detected by electron paramagnetic resonance (EPR) technique. It is suggested that DBNBS may bind to the apoprotein B100 and lipids of LDL by a lysine-independent mechanism resulting in increased recognition and degradation by macrophages. The present work offers a novel approach for rapid modification of LDL.

Benzenesulfonates↗

PubMed1991

Analysis of the monocyte chemotactic response to lysophosphatidylcholine: role of lysophospholipase C.

Previously, we reported that lysophosphatidylcholine (lyso-PtdCho), a component of oxidized low-density lipoprotein, was a monocyte chemoattractant (M.T. Quinn et al. (1988) Proc. Natl. Acad. Sci. USA 85, 2805-2809). Monocyte chemotaxis was also stimulated by lyso-platelet activating factor but not by platelet activating factor itself. In the present studies, we used other analogs of lyso-PtdCho to determine structural and metabolic features required for chemotactic activity. Although both D- and L-lyso-PtdCho stimulated chemotaxis, suggesting a lack of stereospecificity, studies using propanediol and ethanediol analogs of lyso-PtdCho suggested that a free hydroxyl moiety or an ester-linked fatty acid vicinal to the phosphocholine group of the lysophospholipid was required for the expression of activity. Incubation of [3H]choline-labeled lyso-PtdCho with monocytes resulted in the formation of labeled PtdCho, glycerophosphocholine (GPC), phosphocholine, and free choline, while resident peritoneal macrophages, cells which we show do not respond chemotactically to lyso-PtdCho, metabolized the labeled substrate to generate only labeled PtdCho and GPC; no labeled phosphocholine was found, suggesting a possible role for lysophospholipase C activity in the monocyte chemotactic response. Although monoacylglycerol, the product of lysophospholipase C hydrolysis of lyso-PtdCho, was not chemotactic for monocytes, diacylglycerol demonstrated chemotactic activity, suggesting that the subsequent acylation to diacylglycerol may be involved in the monocyte chemotactic response to lyso-PtdCho. Indeed, monocytes incorporated [3H]glycerol from [3H]glycerol-labeled lyso-PtdCho into di- and triacylglycerol. Based on these results, a model is proposed whereby the monocyte chemotactic response to lyso-PtdCho involves a sequence of metabolic steps which includes hydrolysis of lyso-PtdCho to monoacylglycerol and phosphocholine by lysophospholipase C followed by acylation of monoacylglycerol to diacylglycerol. Diacylglycerol would then act as an intracellular second messenger that could activate or facilitate the chemotactic response.

Animals↗