Anna Baoutina

Regulation of serum-induced lipid accumulation in human monocyte-derived macrophages by interferon-γ. Correlations with apolipoprotein E production, lipoprotein lipase activity and LDL receptor-related protein expression

The demonstration of lipid loaded macrophages in atherosclerotic tissue has led to the development of in vitro systems to elucidate the mechanisms involved in lipid accumulation. Here we have characterised the changes which occur in human monocyte-derived macrophage (MDM) lipids during culture in either human serum (HS) or foetal calf serum (FCS). MDM cultured in HS were rapidly converted to lipid filled foam cells, as assessed using HPLC analysis and oil red-O staining and compared with the same cells grown in FCS. However, the lipids which accumulated were predominantly triglycerides with smaller amounts of unesterified cholesterol (UC) and only traces of cholesteryl esters (CE). alpha-Tocopherol (alpha-TocH) was present at higher levels in MDM cultured in HS compared to the same cells grown in FCS. MDM lipid accumulation was dependent on the triglyceride-rich lipoprotein (TGRL) fraction of human serum; accordingly, supplementation of FCS with human TGRL also induced MDM lipid accumulation. The relationships between cellular lipid accumulation and secretion of apolipoprotein E (apo E) and lipoprotein lipase (LPL) as well as expression of the low density lipoprotein receptor-related protein (LRP) were also examined. MDM lipid accumulation was associated with increased apo E secretion but did not alter extracellular LPL activity. The lipid accumulation which was induced by HS was potently inhibited (but not reserved) by the inflammatory cytokine interferon-gamma (IFN gamma), and this was associated with decreased apo E production, LPL secretion and expression of LRP. These studies reveal striking differences in the lipid composition of MDM cultured in either HS or FCS, and indicate that oil red-O staining is not necessarily associated with cholesteryl ester accumulation in human macrophages. Furthermore, the effect that serum-induced lipid accumulation has on the specific MDM functions studied should be appreciated when developing in vitro macrophage models.

Comparative time-courses of copper-ion-mediated protein and lipid oxidation in low-density lipoprotein

Free radicals damage both lipids and proteins and evidence has accumulated for the presence of both oxidised lipids and proteins in aged tissue samples as well as those from a variety of pathologies including atherosclerosis, diabetes, and Parkinsons disease. Oxidation of the protein and lipid moieties of low-density lipoprotein is of particular interest due to its potential role in the unregulated uptake of lipids and cholesterol by macrophages; this may contribute to the initial stage of foam cell formation in atherosclerosis. In the study reported here, we examined the comparative time-courses of lipid and protein oxidation during copper-ion-mediated oxidation of low-density lipoprotein. We show that there is an early, lipid-mediated loss of 40-50% of the Trp residues of the apoB100 protein. There is no comparable loss over an identical period during the copper-ion-mediated oxidation of lipid-free BSA. Concomitant with Trp loss, the antioxidant alpha-tocopherol is consumed with subsequent extensive lipid peroxidation. Further changes to the protein, including the copper-ion-dependent 3.5-fold increase in 3,4-dihydroxyphenylalanine and the copper-ion-independent 3-5-fold increase in o-tyrosine, oxidation products of Tyr and Phe, respectively, only occur after maximal lipid peroxidation. Long incubation periods result in depletion of 3,4-dihydroxyphenylalanine, presumably reflecting further oxidative changes. Overall, copper-ion-mediated oxidation of LDL appears to proceed initially by lipid radical-dependent processes, even though some of the earliest detectable changes occur on the apoB100 protein. This is followed by extensive lipid peroxidation and subsequent additional oxidation of aromatic residues on apoB100, though it is not yet clear whether this late protein oxidation is lipid-dependent or occurs as a result of direct radical attack.

Trans-plasma membrane electron transport induces macrophage-mediated low density lipoprotein oxidation

Oxidation of low‐density lipoprotein (LDL) in the arterial intima has been implicated in atherogenesis. The exact mechanism of this oxidation, however, remains unclear. In the present study we investigated the role of the trans‐plasma membrane electron transport (TPMET) system of macrophages in cell‐mediated LDL oxidation. We demonstrated that TPMET of mouse and human macrophages, measured as reduction of the impermeable oxidant ferricyanide (FEC), was enhanced strongly by loading macrophages with ascorbate (Asc). This appoach also enhanced the ability of cells to oxidize LDL in Hams F‐10 medium. The stimulatory effect of Ascenrichment of macrophages on both processes was not due to release of Asc or other reductants into the medium, as demonstrated by the lack of the effects of ascorbate oxidase and the inhibitors of cellular Asc‐recycling on these processes. This conclusion is also supported by experiments in which we analyzed the amounts of Asc and DHA secreted by cells and tested their effect of LDL oxidation, and also measured the ability of cell‐conditioned medium from control and Asc‐enriched cells to reduce FEC. We conclude that TPMET is largely responsible for macrophage‐mediated LDL oxidation by means of its capacity to reduce extracellular transition metals.

Macrophages Can Decrease the Level of Cholesteryl Ester Hydroperoxides in Low Density Lipoprotein

Murine and human macrophages rapidly decreased the level of cholesteryl ester hydroperoxides in low density lipoprotein (LDL) when cultured in media non-permissive for LDL oxidation. This process was proportional to cell number but could not be attributed to the net lipoprotein uptake. Macrophage-mediated loss of lipid hydroperoxides in LDL appears to be metal ion-independent. Degradation of cholesteryl linoleate hydroperoxides was accompanied by accumulation of the corresponding hydroxide as the major product and cholesteryl keto-octadecadienoate as a minor product, although taken together these products could not completely account for the hydroperoxide consumption. Cell-conditioned medium possessed a similar capacity to remove lipid hydroperoxides as seen with cellular monolayers, suggesting that the activity is not an integral component of the cell but is secreted from it. The activity of cell-conditioned medium to lower the level of LDL lipid hydroperoxides is associated with its high molecular weight fraction and is modulated by the availability of free thiol groups. Cell-mediated loss of LDL cholesteryl ester hydroperoxides is facilitated by the presence of α-tocopherol in the lipoprotein. Together with our earlier reports on the ability of macrophages to remove peroxides rapidly from oxidized amino acids, peptides, and proteins as well as to clear selectively cholesterol 7-β-hydroperoxide, results presented in this paper provide evidence of a potential protective activity of the cell against further LDL oxidation by removing reactive peroxide groups in the lipoprotein.

Antioxidant Properties of Macrophages Toward Low-Density Lipoprotein

Oxidative modification of low-density lipoprotein (LDL) has been implicated in atherosclerosis. Intensive scientific efforts over the last two decades have focused on the elucidation of the mechanisms by which LDL is oxidized in vivo. A wealth of in vitro studies has demonstrated that the cell types present in atherosclerotic lesions, including monocyte/macrophages, quantitatively one of the most important cell types in plaque development, promote LDL oxidation. The mechanisms of cellular prooxidant activities have been extensively investigated. Fewer studies have addressed possible protective properties of the cells in LDL oxidation. This review summarizes recent observations of antioxidant, and potentially antiatherogenic, activities of macrophages toward LDL, including macrophage-mediated detoxification of lipid and protein hydroperoxides, metal sequestration and the generation of compounds with antioxidant properties. These activities could contribute to the net effect of macrophages on deleterious LDL oxidation and to the complex role of these cells in lesion development.

Transplasma membrane redox activity of monocytes/macrophages.

Abstract Oxidation of low density lipoprotein in the arterial intima has been implicated in atherogenesis. Numerous studies have shown that various cells of the arterial wall, including macrophages, are able to oxidatively modify LDL in vitro. Although the exact mechanism of macrophage-mediated LDL oxidation in vitro remains unclear, it is generally accepted that it occurs via a transition metal-dependent process. Recently, it has been demonstrated that macrophages are able to reduce extracellular copper and iron, and the contribution to this reduction of a transplasma membrane electron transport (TPMET) system of the cells has been suggested.1 In the present paper, we investigate the presence of a TPMET system in monocytes/ macrophages and ways to manipulate its activity. The establishment of ways to change the expression or activity of the TPMET system in these cells could provide convenient tool to further investigate the possible correlation between cellular transmembrane electron transport and the ability of cells to oxidise LDL.