Hypoxia regulates the production and activity of glucose transporter-1 and indoleamine 2,3-dioxygenase in monocyte-derived endothelial-like cells: possible relevance to infantile haemangioma pathogenesis.

BACKGROUND: Infantile haemangioma (IH) may present as a precursor area of pallor prior to the initial proliferative phase, which implies that the early lesion may be hypoxic. OBJECTIVES: To examine the effect of hypoxia on the expression and activity of two key molecular markers of IH, glucose transporter-1 (GLUT1) and indoleamine 2,3-dioxygenase (IDO). METHODS: IH endothelial cells express both haematopoietic and endothelial cell markers. CD14+ monocyte-derived endothelial-like cells have been employed in the study of IH and is the cell type used in this study. RESULTS: GLUT1 transcript, protein and activity levels were strongly induced by hypoxia and remained elevated following 2 days of normoxic recovery. IDO transcript levels were not affected by hypoxia, although IDO protein level was reduced fivefold and IDO activity >100-fold following 2 days of hypoxia. The protein and activity levels returned to normal following 2 days of normoxic recovery. CONCLUSIONS: The findings link the tissue hypoxia that precedes lesion development and the expression and/or activity of two key IH proteins. The early hypoxic insult may contribute to the elevated GLUT1 levels in IH lesions, while the very low IDO activity during the hypoxic phase may promote activation of immune cells in the lesion, which release cytokines that trigger IDO expression and activity and entry into the proliferative phase. Interestingly, IH lesion development shares some common features with ischaemia-reperfusion injury.

Lipid oxidation in atherogenesis: an overview.

The 'oxidation theory' for atherosclerosis proposes that lipid and/or protein oxidation products are responsible for lesion formation/development. The major target for oxidation is suggested to be intimal low-density lipoprotein. This idea was stimulated by the pro-atherogenic properties of in vitro oxidized lipoproteins, such as stimulation of chemotaxis and sterol accumulation in macrophages, adhesion molecule expression on endothelial cells and apoptosis of several cell types. It was supported by detection of oxidation products in lesion lipoproteins, although these are (in general) less heavily oxidized and their biological activity is less rigorously defined than for their in vitro oxidized counterparts. Lesion lipids contain products generated by both enzymic and non-enzymic oxidation reactions; the majority are generated non-enzymically. The type and source of oxidant involved has been the subject of much speculation and is not resolved. The oxidation theory predicts that appropriate antioxidants will protect against atherosclerosis. Vitamin E has been used in several animal and human studies, but to date has shown little evidence of anti-atherosclerotic potential. However, lack of knowledge of the oxidant(s) driving lesion oxidation and the complexity of the anti- and pro-oxidant properties of vitamin E may explain its disappointing track record to date. These subjects require more rigorous study before the oxidation theory can be fairly tested.

Endothelin-1 plus oxidized low-density lipoprotein, but neither alone, increase human monocyte adhesion to endothelial cells.

Endothelin-1 is a potent vasoconstrictor and mitogenic peptide that is implicated in the atherosclerosis of apolipoprotein E-deficient mice and may promote atherogenesis in humans. We hypothesized that endothelin-1 might promote the adhesion of monocytes to endothelial cells, a key early event in atherosclerosis. We investigated the adhesion of primary human monocytes (isolated by elutriation) to human umbilical vein endothelial cell cultures after incubation with endothelin-1 (0.1 and 0.01 nM; approximately physiological concentrations), copper-oxidized low-density lipoprotein (LDL) (0.1 mg/ml) and a combination of the two. After a 4 h incubation with 0.1 or 0.01 nM endothelin-1 combined with oxidized LDL, adhesion was increased to 120+/-4% (P<0.001 compared with control) and 118+/-4% (P<0.002) respectively, whereas neither substance alone increased adhesion (92-104% of control values; not significant). Neither endothelin receptor A blockade nor co-incubation with anti-fibronectin antibody inhibited the pro-adhesive effects of endothelin-1 plus oxidized LDL (115+/-7% and 115+/-3% of control compared with 120+/-4% respectively; not significant). Endothelial cell expression of intercellular adhesion molecule-1, vascular adhesion molecule-1 and E-selectin were unchanged throughout the experiment. Therefore physiological concentrations of endothelin-1 and oxidized LDL may act synergistically to increase the adhesion of human monocytes to endothelial cells, contributing in part to the observed pro-atherogenic effects of endothelin-1.

Inhibition of cholesterol efflux by 7-ketocholesterol: comparison between cells, plasma membrane vesicles, and liposomes as cholesterol donors.

Cholesterol removal from lipid-loaded macrophages is an important, potentially antiatherogenic process, and we have previously shown that an oxysterol, 7-ketocholesterol (7K), can impair efflux to lipid-free apoprotein A-1 (apoA-1). This publication investigates whether incorporation of 7K into membranes could account for this impairment of cholesterol efflux. Cholesterol efflux was studied from lipoprotein-loaded THP-1 cells, from plasma membrane vesicles obtained from these cells, and from artificial, protein-free liposomes. Impairment of cholesterol efflux by 7K was observed for all cholesterol donor systems whether measured as decline in cholesterol removal rates or as the percentage mass of total cellular cholesterol exported. 7-Ketocholesterol itself was not removed by apoA-1 from any of the cholesterol donor systems. Increasing membrane cholesterol content increased the rate of cholesterol removal by apoA-1 (as seen with plasma membrane vesicles), the quantity of cholesterol removed at equilibrium (liposomes), or both (whole cells). Although the minimum inhibitory 7K concentrations varied between the cholesterol donor systems, 7K inhibited cholesterol efflux in all systems. It was concluded that 7K induces alteration in membranes which decreased the efficiency of cholesterol efflux and the quantity of removed cholesterol induced by apoA-1. As cell membrane proteins are not essential for cholesterol efflux in these systems, the impairment of such by 7K suggests that its effect on membrane lipid composition and its structure are key regulatory elements in this efflux process.

Sex-related differences in the regulation of macrophage cholesterol metabolism.

Men have an earlier onset and higher incidence of coronary heart disease than women, independent of environmental risk factor exposure. As a consequence, there has been considerable interest in the potential role of sex hormones in atherogenesis. An emerging body of evidence suggests that sex-specific tissue and cellular characteristics may mediate sex-specific responses to a variety of stimuli. Recent studies have shown that oestrogen, progesterone and androgens all regulate processes integral to human macrophage foam cell formation, a key event in atherogenesis, in a sex-specific manner; findings that may have important implications for understanding the sex gap in atherosclerosis. Physiological levels of 17beta-estradiol and progesterone are both associated with a female-specific reduction in cholesteryl ester accumulation in human macrophages. By contrast, androgens increase cholesteryl ester formation in male but not in female donor human macrophages. This review summarizes current data concerning the sex-specific effects of sex hormones on processes important to macrophage foam cell formation and the basic mechanisms responsible for the sex specificity of such effects. Future research in this promising field may eventually lead to the novel concept of 'sex-specific' treatments directed at inhibiting atherogenesis.

A kinetic model to evaluate cholesterol efflux from THP-1 macrophages to apolipoprotein A-1.

The kinetics (0 to 3 h) of cholesterol efflux to delipidated apolipoprotein A-1 were investigated, and the experimental data were best fitted to a mathematical model that involves two independent pathways of cholesterol efflux. The first pathway with a rate constant of 4.6 h(-1) is fast but removes only 3-5% of total cholesterol. After preconditioning apoA-1, it was found that this pathway remains, and hence it is a property of the cholesterol-loaded cells rather than due to modification on the apolipoprotein. This fast initial efflux does not seem to contribute to cholesterol efflux at later stages (>1 h) where a second pathway predominates. However, the fast initial efflux pool can be restored if apoA-1 is withdrawn. The second slower pathway (k(membrane--media) = 0.79 h(-1)) is associated with cholesterol ester hydrolysis whose rate constant could be experimentally verified (k(cal) = 0.43, k(exp) = 0.38 +/- 0.05). The model suggests that two different plasma membrane domains are involved in the two pathways. Loading of the cells with an oxysterol, 7-ketocholesterol (7K), inhibits efflux from both pathways. The model predicts that 7K decreases the initial efflux by decreasing the available cholesterol (by possibly affecting lipid packing), while all rate constants in the second pathway are decreased. In conclusion, the kinetic model suggests that cholesterol efflux to apoA-1 is a two-step process. In the first step, some of the plasma membrane cholesterol contributes to a fast initial efflux (possibly from lipid rafts) and leads to a second pathway that mobilizes intracellular cholesterol mobilization.

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.

Enhancement of macrophage survival and DNA synthesis by oxidized-low-density-lipoprotein (LDL)-derived lipids and by aggregates of lightly oxidized LDL.

Human atherosclerotic plaque contains a partially characterized range of normal and oxidized lipids formed mainly from free and esterified cholesterol and phospholipids, some of which can be located in macrophage-derived "foam" cells. Oxidation of low-density lipoprotein (LDL) is often considered as an important event leading to subsequent foam-cell development, which may also include enhanced cell survival and/or proliferation. The active component(s) in oxidized LDL (ox.LDL) causing macrophage proliferation is debated. We report here that the lipid component of ox.LDL can promote macrophage survival and DNA synthesis, the latter response showing a synergistic effect in the presence of low concentrations of macrophage colony-stimulating factor. 7-Ketocholesterol showed some stimulation of macrophage DNA synthesis whereas hypochlorite-oxidized (i.e. apolipoprotein B-oxidized) LDL did not. Plaque-derived lipids could enhance macrophage survival. It has not been proven that LDL in lesions is oxidized sufficiently to be the dominant source of sterols in vivo or to be able to induce macrophage growth in vitro or in vivo; it has been suggested that aggregation of modified LDL in vivo is an important step in the deposition of intracellular lipid. We found that aggregation of lightly oxidized LDL potentiated dramatically its ability to stimulate macrophage DNA synthesis, indicating that extensive oxidation of LDL is not required for this response in vitro and perhaps in vivo.

Comparison of macrophage responses to oxidized low-density lipoprotein and macrophage colony-stimulating factor (M-CSF or CSF-1).

Modification of low-density lipoprotein (LDL), for example by oxidation, could be involved in foam cell formation and proliferation observed in atherosclerotic lesions. Macrophage colony-stimulating factor (CSF-1 or M-CSF) has been implicated in foam cell development. It has been reported previously that oxidized LDL (ox.LDL) and CSF-1 synergistically stimulate DNA synthesis in murine bone-marrow-derived macrophages (BMM). The critical signal-transduction cascades responsible for the proliferative response to ox.LDL, as well as their relationship to those mediating CSF-1 action, are unknown. We report here that ox.LDL stimulated extracellular signal-regulated protein kinase (ERK)-1, ERK-2 and phosphoinositide 3-kinase activities in BMM but to a weaker extent than optimal CSF-1 concentrations at the time points examined. Inhibitor studies suggested at least a partial role for these kinases, as well as p70 S6-kinase, in ox.LDL-induced macrophage survival and DNA synthesis. For the DNA synthesis response to CSF-1, the degree of inhibition by PD98059, wortmannin and rapamycin was significant at low CSF-1 concentrations but was reduced as the CSF-1 dose increased. Using BMM from CSF-1-deficient mice (op/op) and a neutralizing antibody approach, we found no evidence for an essential role for endogenous CSF-1 in ox.LDL-mediated survival or DNA synthesis; likewise, with the same approaches, no evidence was obtained for an essential role for endogenous granulocyte/macrophage-CSF in ox.LDL-mediated macrophage survival and, in contrast with the literature, ox.LDL-induced macrophage DNA synthesis.

Metabolism of oxidized LDL by macrophages.

Oxidation products of lipids and proteins are found in atherosclerotic plaque and in macrophage foam cells. Macrophages avidly endocytose in-vitro oxidized LDL and accumulate sterols. What is the evidence that such a process is involved in in-vivo foam cell formation? The present review surveys current knowledge on the metabolism of oxidized LDL by macrophages, and the types, amounts and location of oxidation products that accumulate in these cells. Comparable studies of lesion lipoproteins and foam cells indicate that limited extracellular lipoprotein oxidation, perhaps followed by more extensive intracellular oxidation subsequent to uptake by macrophages, is a more likely scenario in vivo.

Apolipoprotein A-I, cyclodextrins and liposomes as potential drugs for the reversal of atherosclerosis. A review.

Several studies have revealed that high-density lipoprotein (HDL) is the most reliable predictor for susceptibility to cardiovascular disease. Since apolipoprotein A-I (apoA-I) is the major protein of HDL, it is worthwhile evaluating the potential of this protein to reduce the lipid burden of lesions observed in the clinic. Indeed, apoA-I is used extensively in cell culture to induce cholesterol efflux. However, while there is a large body of data emanating from in-vitro and cell-culture studies with apoA-I, little animal data and scant clinical trials examining the potential of this apolipoprotein to induce cholesterol (and other lipid) efflux exists. Importantly, the effects of oxysterols, such as 7-ketocholesterol (7KC), on cholesterol and other lipid efflux by apoA-I needs to be investigated in any attempt to utilise apoA-I as an agent to stimulate efflux of lipids. Lessons may be learnt from studies with other lipid acceptors such as cyclodextrins and phospholipid vesicles (PLVs, liposomes), by combination with other effluxing agents, by remodelling the protein structure of the apolipoprotein, or by altering the composition of the lipoprotein intended for administration in-vivo. Akin to any other drug, the usage of this apolipoprotein in a therapeutic context has to follow the traditional sequence of events, namely an evaluation of the biodistribution, safety and dose-response of the protein in animal trials in advance of clinical trials. Mass production of the apolipoprotein is now a simple process due to the advent of recombinant DNA technology. This review also considers the potential of cyclodextrins and PLVs for use in inducing reverse cholesterol transport in-vivo. Finally, the potential of cyclodextrins as delivery agents for nucleic acid-based constructs such as oligonucleotides and plasmids is discussed.

HMG CoA reductase inhibition reduces sarcolemmal Na(+)-K(+) pump density.

OBJECTIVES: HMG CoA reductase inhibitors reduce cellular availability of mevalonate, a precursor in cholesterol synthesis. Since the cholesterol content of cell membranes is an important determinant of Na(+)-K(+) pump function we speculated that treatment with HMG CoA reductase inhibitors affects Na(+)-K(+) pump activity. METHODS: We treated rabbits and rats for 2 weeks with the HMG CoA reductase inhibitor lovastatin and measured Na(+)-K(+) pump current (I(p)) in isolated rabbit cardiac myocytes using the whole cell patch-clamp technique, K-dependent p-nitrophenyl phosphatase (p-NPPase) activity in crude myocardial and skeletal muscle homogenates, and vanadate-facilitated 3H-ouabain binding in intact skeletal muscle samples from rats. RESULTS: Treatment with lovastatin caused statistically significant reductions in I(p), myocardial and skeletal muscle K-dependent p-NPPase activity and 3H-ouabain binding in the myocardium and skeletal muscle. The lovastatin-induced decrease in I(p) was eliminated by parenteral co-administration of mevalonate. However, this was not related to cardiac cholesterol content. CONCLUSIONS: Treatment with lovastatin reduces Na(+)-K(+) pump activity and abundance in rabbit and rat sarcolemma.

Regulation of apolipoprotein E production in macrophages (review).

Apolipoprotein E (apo E), a 34 kDa component of lipoproteins produced by the liver and in circulating macrophages, plays a critical role in the reverse transport of cholesterol to the liver via the circulation. Cholesterol-rich macrophages (macrophage foam cells) are a major cell type in human atherosclerotic lesions. Apo E deficiency in mice leads to the formation of atherosclerotic lesions. Conversely, macrophage-specific expression of apo E in these deficient mice can reduce the extent of atherosclerosis. These observations, together with the anti-inflammatory and anti-proliferative properties of Apo E, demonstrate an atheroprotective role for the apolipoprotein. Agents that regulate macrophage metabolism are also able to modulate apo E expression. Sterol loading, for example, enhances apo E synthesis and secretion. Additionally, exposure of macrophage foam cells to cholesterol acceptors such as apo A-1, the protein component of high density lipoprotein, further enhance apo E secretion. Cytokines can have a negative regulatory effect on apo E production in macrophages. Apo E expression is controlled at the transcriptional, post-transcriptional and post-translational level. Here, we review the cellular and molecular mechanisms modulating apo E synthesis and secretion in macrophages.

Sterol 27-hydroxylase acts on 7-ketocholesterol in human atherosclerotic lesions and macrophages in culture.

27-Hydroxycholesterol (27OH) is the major oxysterol in human atherosclerotic lesions, followed by 7-ketocholesterol (7K). Whereas 7K probably originates nonenzymically, 27OH arises by the action of sterol 27-hydroxylase, a cytochrome P450 enzyme expressed at particularly high levels in the macrophage and proposed to represent an important pathway by which macrophages eliminate excess cholesterol. We hypothesized and here show that 27-hydroxylated 7-ketocholesterol (270H-7K) is present in human lesions, probably generated by the action of sterol 27-hydroxylase on 7K. Moreover, [(3)H]27OH-7K was produced by human monocyte-derived macrophages (HMDMs) supplied with [(3)H]7K but not in HMDMs from a patient with cerebrotendinous xanthomatosis (CTX) shown to have a splice-junction mutation of sterol 27-hydroxylase. Whereas [(3)H]27OH-7K was predominantly secreted into the medium, [(3)H]-27OH formed from [(3)H]-cholesterol was mostly cell-associated. The majority of supplied [(3)H]7K was metabolized beyond 27OH-7K to aqueous-soluble products (apparently bile acids derived from the sterol 27-hydroxylase pathway). Metabolism to aqueous-soluble products was ablated by a sterol 27-hydroxylase inhibitor and absent in CTX cells. Sterol 27-hydroxylase therefore appears to represent an important pathway by which macrophages eliminate not only cholesterol but also oxysterols such as 7K. The fact that 7K (and cholesterol) still accumulates in lesions and foam cells indicates that this pathway may be perturbed in atherosclerosis and affords a new opportunity for the development of therapeutic strategies to regress atherosclerotic lesions.

Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells. Accumulation of oxidized esters in lysosomes.

Cholesterol- and cholesteryl ester-rich macrophage foam cells, characteristic of atherosclerotic lesions, are often generated in vitro using oxidized low density lipoprotein (OxLDL). However, relatively little is known of the nature and extent of sterol deposition in these cells or of its relationship to the foam cells formed in atherosclerotic lesions. The purpose of this study was to examine the content and cellular processing of sterols in OxLDL-loaded macrophages, and to compare this with macrophages loaded with acetylated LDL (AcLDL; cholesteryl ester-loaded cells containing no oxidized lipids) or 7-ketocholesterol-enriched acetylated LDL (7KCAcLDL; cholesteryl ester-loaded cells selectively supplemented with 7-ketocholesterol (7KC), the major oxysterol present in OxLDL). Both cholesterol and 7KC and their esters were measured in macrophages after uptake of these modified lipoproteins. Oxysterols comprised up to 50% of total sterol content of OxLDL-loaded cells. Unesterified 7KC and cholesterol partitioned into cell membranes, with no evidence of retention of either free sterol within lysosomes. The cells also contained cytosolic, ACAT-derived, cholesteryl and 7-ketocholesteryl esters. The proportion of free cholesterol and 7KC esterified by ACAT was 10-fold less in OxLDL-loaded cells than in AcLDL or 7KCAcLDL-loaded cells. This poor esterification rate in OxLDL-loaded cells was partly caused by fatty acid limitation. OxLDL-loaded macrophages also contained large (approximately 40-50% total cell sterol content) pools of oxidized esters, containing cholesterol or 7KC esterified to oxidized fatty acids. These were insensitive to ACAT inhibition, very stable and located in lysosomes, indicating resistance to lysosomal esterases. Macrophages loaded with OxLDL do not accumulate free sterols in their lysosomal compartment, but do accumulate lysosomal deposits of OxLDL-derived cholesterol and 7-ketocholesterol esterified to oxidized fatty acids. The presence of similar deposits in lesion foam cells would represent a pool of sterols that is particularly resistant to removal.

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 alpha-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-beta-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.

Androgen receptor expression is greater in macrophages from male than from female donors. A sex difference with implications for atherogenesis.

BACKGROUND: Male sex is an independent risk factor for the extent and severity of atherosclerosis. The influence of androgens on foam cell formation, a key event in atherogenesis, has not yet been investigated. METHODS AND RESULTS: Primary human monocytes were allowed to differentiate into macrophages. RNA was then extracted from healthy male-donor (n=8) and premenopausal female-donor (n=8) macrophages, and message for the androgen receptor (AR) was examined by RT-PCR. There was a significantly higher level of AR mRNA in macrophages isolated from men than in those from women (0.64+/-0.06 versus 0.15+/-0.02 amol/microgram total RNA; P<0.001). AR mRNA levels were similar in macrophages from postmenopausal and premenopausal women (P=0.16). The functional consequence of this sex difference was then explored. Lipid-loading studies were performed on male (n=9) macrophages treated with the androgen dihydrotestosterone (DHT) and/or the AR antagonist hydroxyflutamide. These showed that DHT caused a dose-dependent and receptor-mediated increase in macrophage cholesteryl ester content (109+/-10%, 117+/-3%, and 120+/-4% for 4, 40, and 400 nmol/L DHT, respectively, as a percentage of control, P=0.002; 95+/-8% for DHT with hydroxyflutamide, P=0.58 versus controls). By contrast, there was no significant effect of androgen on lipid loading in female-donor macrophages (P>0.2 versus controls). CONCLUSIONS: Sex differences in androgen-mediated macrophage lipid loading may contribute to the greater prevalence and severity of atherosclerosis in men.