Role of superoxide in endothelial-cell modification of low-density lipoproteins

Cultured endothelial cells and arterial smooth muscle cells have been shown to modify LDL in a way that leads to rapid uptake by macrophages. Previous studies have demonstrated that this modification involves free radical peroxidation of LDL, and that the role of the cells was to accelerate oxidation under conditions where it otherwise would occur slowly. The objective of the present study was to determine whether the modification was mediated by oxygen-derived free radicals, and whether the ability of a given cell type of line to modify LDL was related to its secretion rate of O2- or H2O2. The results showed that modification required the presence of oxygen, and could be specifically inhibited by superoxide dismutase but not by catalase or by mannitol, a hydroxyl radical scavenger. Rabbit aortic endothelial cells, rabbit arterial smooth muscle cells, monkey arterial smooth muscle cells and human skin fibroblasts were all found to modify LDL, and all of these cell types generated more O2- (superoxide dismutase-inhibitable cytochrome c reduction) than a line of bovine aortic endothelial cells that did not modify LDL. The content of superoxide dismutase and catalase was higher in bovine aortic endothelial cells than in the cell lines that modified LDL, but glutathione peroxidase levels were not different. It was concluded that cells that were capable of modifying LDL produced superoxide or a substance that could be converted to superoxide in the medium, and that superoxide was an important, though possibly indirect, mediator of the modification of LDL by cells.     

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

Pigeon aortic smooth muscle cells lack a functional low density lipoprotein receptor pathway

The low density lipoprotein (LDL) receptor pathway was studied in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons and compared with rhesus monkey cells whose LDL receptor pathway has been previously characterized. Pigeon LDL was bound with high affinity in a saturable manner to both pigeon and monkey aortic smooth muscle cells. The kinetics of binding were different, however. LDL binding to pigeon cells exhibited positive cooperativity at low LDL concentrations and at least two classes of binding sites. The same pigeon LDL bound to monkey cells in a manner consistent with a single class of binding sites. Thus, these differences were a property of the pigeon cells and not the result of differences in the LDL. On the average, pigeon cells bound less than 50% the amount of LDL as monkey cells. Despite the surface binding to pigeon cells, little of the LDL was internalized, whereas pigeon LDL was actively internalized by monkey cells. Consistent with this observation, chloroquine and leupeptin had no effect on accumulation of LDL or on LDL degradation by pigeon cells, and incubation of pigeon cells with LDL produced no increase in cellular cholesteryl ester content. Binding of LDL to pigeon cells also differed from that of monkey cells by being unaffected by pretreatment with the proteolytic enzyme pronase, and by not requiring calcium. Binding was not specific for LDL since acetyl-LDL, and to a lesser degree HDL, were able to compete for LDL binding. Incubation with lipoprotein-deficient serum decreased LDL binding in pigeon cells while 25-OH cholesterol caused an increase in binding; both effects are opposite of that seen with the same LDL in mammalian cells. Preincubation with LDL or cholesterol dissolved in ethanol were without effect on LDL binding in pigeon cells, even though they produced significant increases in cellular free cholesterol content. In spite of the failure to internalize LDL, there was considerable degradation of LDL. This apparently occurred on the cell surface rather than by internalization and degradation within the lysosomes as occurs in mammalian cells. The functional significance of LDL binding to pigeon smooth muscle cells is unclear. The characteristics of binding resemble that of a nonspecific lipoprotein receptor referred to by others as the "lipoprotein receptor" or the "EDTA-insensitive receptor." It is apparent, however, that White Carneau pigeon aortic smooth muscle cells lack a functional LDL receptor pathway and in this way resemble cells from human beings with homozygous familial hypercholesterolemia or from Watanabe rabbits.     

Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.     

Expression of the acetyl low density lipoprotein receptor by rabbit fibroblasts and smooth muscle cells. Up-regulation by phorbol esters

The acetyl low density lipoprotein (LDL), or scavenger, receptor, which binds modified forms of LDL, was thought to be expressed only on macrophages and endothelial cells. We demonstrate that rabbit fibroblasts and smooth muscle cells bind, internalize, and degrade acetoacetylated LDL, a ligand for the acetyl LDL receptor. Degradation is specific in that unlabeled acetoacetylated LDL and fucoidin, a known competitor for binding to the acetyl LDL receptor, are effective competitors, while native LDL is not. The acetyl LDL receptor on these cells is readily regulated. Higher levels of degradation are observed in cells preincubated with serum than in cells preincubated with plasma. This up-regulation of the acetyl LDL receptor is most likely due to the presence of platelet secretory products in serum since secretion products derived from thrombin-stimulated platelets also cause an increase in degradation. In addition, preincubation of rabbit fibroblasts with phorbol esters results in a 16-20-fold increase in specific degradation. These results indicate that rabbit fibroblasts and smooth muscle cells express the acetyl LDL receptor and that increased receptor expression appears to be mediated through activation of the protein kinase C pathway.     

The effect of hypoxia on uptake and degradation of low density lipoproteins by cultured human arterial smooth muscle cells.

Atheroma have been produced in experimental animal by systemic hypoxia. This study assessed the effects of hypoxia on binding, uptake and degradation of human low density lipoprotein (LDL) by human arterial smooth muscle cells, the cell involved in atherogenesis. The LDL content of the smooth muscle cell grown in the usual conditions (95% air [20% O2], 5% CO2) increased with the incubation time of LDL in the medium (7.5 mug protein/ml of medium); the trypsin releasable LDL "binding" reached a plateau by 24 h (2.2 +/- 1.3 [x +/- S.D.]) ng/mug LDL protein added per 10(6) cells whereas the LDL in the cell after trypsinization ("net uptake") continued to increase up to 48 h (6.5 +/- 4.6 ng/mug LDL protein added per 10(6) cells at 48 h). LDL protein degradation increases rapidly between 7 and 48 h (10.4 ng/mug LDL protein added per 10(6) cells at 24 h) after an initial delay of approximately 7 h. Smooth muscle cells grown under hypoxic conditions (5%02) had similar LDL "binding " but showed increased "net uptake" (10.7 +/- 4.8 ng/mug LDL protein added per 10(6) cells) and a 36 +/- 13% decrease in degradation (p less than 0.05; n =8). The impaired degradation of lipoprotein by smooth muscle cells may, in part, explain the role of hypoxia in atherogenesis.     

Uptake and degradation of low density lipoproteins in atherosclerotic rabbit aorta: role of local LDL modification

The uptake of native and modified low density lipoprotein (LDL) in foam cells in atherosclerotic tissue was studied in an in vitro perfusion system for rabbit aorta. Experimental atherosclerosis was induced in rabbits by a combination of cholesterol feeding and mechanical injury. The aorta was perfused in an incubation chamber. A trace-label, radioiodinated tyramine-cellobiose, was used to study cellular uptake of lipoproteins. After perfusion, the tissue was digested and cells were isolated by centrifugation in a density gradient. About 40 times more LDL per cell was accumulated in the foam cell fraction than in the smooth muscle cell fraction. When the cellular uptake of LDL and acetylated LDL (AcLDL) was compared, about 4 times more AcLDL than LDL was taken up by the foam cells, suggesting that the scavenger receptor is expressed in these cells. In a competition experiment, the uptake of LDL into foam cells was reduced by 70% when a tenfold excess of AcLDL was added. This experiment suggests that native LDL is taken up by the same mechanism as AcLDL. The accumulation of radiolabeled LDL in plaques and in foam cells was reduced by 30-55% by adding vitamin E (0.1 mg/ml) to the system. These studies show an uptake of LDL by foam cells in the atherosclerotic tissue. Furthermore, these cells seem to express the scavenger receptor. The competition experiment would suggest that native LDL is taken up by the scavenger receptor. The observation that an antioxidant, vitamin E, may decrease this uptake suggests that oxidative modification of LDL is of importance for this process.     

Stimulation of receptor-dependent and receptor-independent pathways of low-density lipoprotein degradation in arterial smooth muscle cells by platelet-derived growth factor

Platelet-derived growth factor (PDGF), a powerful mitogen released by platelets, promoted the degradation of low-density lipoprotein (LDL) by cultured primate arterial smooth muscle cells and human skin fibroblasts by stimulating both receptor-mediated and LDL-receptor-independent uptake of LDL. Stimulation of LDL-receptor-independent LDL uptake and degradation by PDGF was demonstrated in three ways. First, the small amount of LDL that was degraded by LDL-receptor-negative skin fibroblasts was stimulated by PDGF. Second, PDGF led to increased degradation of LDL that had been reductively methylated to prevent its binding to LDL receptors. Third, 125I-labeled LDL degradation was stimulated by PDGF in the presence of high concentrations of unlabeled LDL, i.e., conditions under which the contribution of the LDL receptor to cellular uptake and degradation is reduced. These observations suggest that mitogens, as typified by PDGF, can facilitate the cellular delivery of LDL cholesterol by both LDL-receptor-mediated and non-LDL-receptor-mediated mechanisms to provide exogenous cholesterol for use during cell replication.     

Geldanamycin leads to superoxide formation by enzymatic and non-enzymatic redox cycling. Implications for studies of Hsp90 and endothelial cell nitric-oxide synthase

The ansamycin antibiotic geldanamycin has frequently been used as an inhibitor of heat shock protein 90 (Hsp90), and this agent has been widely employed as a probe to examine the interactions of Hsp90 with endothelial nitric-oxide synthase. Geldanamycin contains a quinone group, which may participate in redox cycling. When geldanamycin was exposed to the flavin-containing enzyme cytochrome P-450 reductase, both semiquinone and superoxide (O(2)(*)(-)) radicals were detected using electron spin resonance. The treatment of endothelial cells with geldanamycin resulted in a dramatic increase in O(2)(*)(-) generation, which was independent of endothelial nitric-oxide synthase, because it was not inhibited by N-nitro-l-arginine methyl ester and also occurred in vascular smooth muscle cells. Diphenylene iodinium inhibited this increase in O(2)(*)(-) by 50%, suggesting that flavin-containing enzymes are involved in geldanamycin-induced O(2)(*)(-) generation. In the absence of cells, geldanamycin directly oxidized ascorbate, consumed oxygen, and produced O(2)(*)(-). Geldanamycin decreased the bioavailable nitric oxide generated by 3,4-dihydrodiazete 1,2-dioxide in smooth muscle cells by 50%, whereas pretreatment with superoxide dismutase inhibited the effect of geldanamycin. These findings demonstrate that geldanamycin generates O(2)(*)(-), which scavenges nitric oxide, leading to loss of its bioavailability. This effect is independent of the inhibition of Hsp90 and indicates that geldanamycin cannot be used as a specific inhibitor of Hsp90. In light of these findings, the studies using geldanamycin as an inhibitor of Hsp90 should be interpreted with caution.     

Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein B by products of fatty acid peroxidation

Uptake of cholesterol-containing lipoproteins by macrophages in the arterial intima is believed to be an important step in the pathogenesis of atherosclerosis. There are a number of possible mechanisms by which macrophages might accumulate cholesterol, and one that has attracted much interest recently involves the uptake of oxidatively modified low density lipoprotein (LDL) via a specific cell surface receptor, termed the scavenger or acetyl-LDL receptor. Previous studies have shown that chemical derivatization of LDL with reagents that result in neutralization of the charge of lysine amino groups also allows recognition by this receptor. As well, it has been shown that oxidation of LDL is accompanied by a decrease in free lysine groups and binding of lipid products to apolipoprotein B. The present studies were done to further characterize the receptor-binding domain on oxidized LDL. It was found that LDL could be modified by incubation with water-soluble products derived from autoxidized unsaturated fatty acids under conditions that inhibited oxidation of the LDL itself. The LDL modified in this way had increased electrophoretic mobility but showed no evidence of the oxidative damage that typifies LDL oxidized by exposure to metal ions. Furthermore, the oxidation product-modified LDL was rapidly degraded by cultured macrophages through the scavenger receptor pathway. Bovine albumin modified by oxidation products also showed greatly accelerated degradation by macrophages. When analyzed by reverse-phase high pressure liquid chromatography, the reactive oxidation products appeared less polar than fatty acids or simple medium-chain aldehydes. When treated with the carbonyl reagent 2,4-dinitrophenylhydrazine, the reactive fractions yielded derivatives, some of which were identified by mass spectrometry as hydrazones of nonenal, heptenal, pentenal, and crotonaldehyde. A series of 2-unsaturated aldehydes (acrolein to 2-nonenal) were all found to modify LDL, but none of these aldehyde-modified LDLs were recognized by the scavenger receptor of macrophages and all were degraded much more slowly by these cells than LDL modified with oxidation products. Furthermore, copper-oxidized LDL had only very slight immunoreactivity toward a panel of antibodies specific for adducts of simple 2-unsaturated aldehydes. Analysis of underivatized autoxidized fatty acids by coupled liquid chromatography/thermospray mass spectrometry revealed compounds with m/z corresponding to M+17, M+31, and 2M+31 in fractions that were capable of modifying LDL. The unoxidized fatty acids showed a dominant peak at M-1. These results indicate that the scavenger receptor of macrophages can recogn     

Effects of ammonium ion derived from bovine endothelial cells upon low density lipoprotein degradation in cultured vascular smooth muscle cells

Low density lipoprotein (LDL) metabolism in bovine arterial smooth muscle cells (SMC) was increased upon exposure to endothelial cell conditioned medium. The mass of LDL degraded in the SMC lysosomal system was increased, and kinetic analysis demonstrated that the rate constant for LDL degradation arising from receptor-mediated endocytosis was unchanged. The effects on LDL metabolism were accompanied by stimulation of DNA synthesis in the SMC. These results are in contrast to reports concerning a porcine endothelial cell system where LDL degradation was inhibited by endothelial-derived NH4+. We show that bovine endothelial cells produce insufficient NH4+ to inhibit LDL degradation and conclude that endothelial cell-derived NH4+ is unlikely to be a factor affecting LDL metabolism in the bovine vascular cell culture system.     

Transforming growth factor-beta up-regulates low density lipoprotein receptor-mediated cholesterol metabolism in vascular smooth muscle cells.

The effects of transforming growth factor-beta (TGF-beta) on low density lipoprotein (LDL) receptor-mediated cholesterol metabolism were evaluated in vascular smooth muscle cells. TGF-beta significantly increased the binding, uptake, and degradation of 125I-LDL. This increase was paralleled by an increase in LDL receptor mRNA steady state levels and an increase in cholesterol esterification. The increase in LDL cholesterol metabolism was independent of proliferation. LDL receptor expression in response to TGF-beta was not affected by coincubation with an antibody against platelet-derived growth factor or by cyclooxygenase inhibitors in arterial smooth muscle cells, suggesting that TGF-beta's effect was not mediated through platelet-derived growth factor or prostaglandins, as demonstrated in other cell systems. However, coincubation with pertussis toxin abrogated the effect of TGF-beta on LDL receptor expression, suggesting that a pertussis toxin-sensitive G-protein may be involved in the signal transduction pathway. These results are discussed in terms of their potential effects on cellular cholesterol trafficking.     

Sialic acid content of human low density lipoproteins affects their interaction with cell receptors and intracellular lipid accumulation.

Low density lipoproteins (LDL) isolated from the plasma of patients with angiographically demonstrable coronary heart disease (CHD) induced accumulation of triglycerides, free cholesterol, and cholesteryl esters in cultured macrophages, smooth muscle cells, and endothelial cells derived from uninvolved intima of human aorta, but not in skin fibroblasts or hepatoma cells. The sialic acid content of LDL from CHD patients was 40-75% lower than that from healthy donors. There was a negative correlation between LDL sialic acid content and the LDL-induced accumulation of total intracellular cholesterol. Neuraminidase treatment of LDL from normal healthy donors produced sialic acid-depleted LDL (Ds-LDL) which was able to stimulate intracellular lipid accumulation. Neuraminidase treatment of LDL from CHD patients further increased its capacity to induce intracellular lipid accumulation. Sialic acid-poor LDL isolated by affinity chromatography of LDL from CHD patients induced a 2- to 4-fold increase of free and esterified cholesterol in human intimal smooth muscle cells. Binding, uptake, and degradation of 125I-labeled Ds-LDL by macrophages and endothelial cells were 1.5- to 2-fold higher than for native LDL. Binding and uptake of Ds-LDL was inhibited 64-93% by the addition of 20-fold excess acetylated LDL (Ac-LDL); in the inverse experiment, the level of inhibition was 35-54%. These data indicate that a sialic acid-poor form of LDL isolated from CHD patients can interact with both native and scavenger LDL receptors. A sialic acid-poor form of LDL may be a naturally occurring ligand that interacts with the scavenger receptor(s) on macrophages and endothelial cells.     

Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes.

The oxygen-derived free radical superoxide anion (.O2-) plays an important role in the pathogenesis of various diseases. Recent demonstrations that .O2- inactivates the potent vasodilator endothelium-derived relaxing factor (EDRF) and that EDRF is probably nitric oxide (NO) suggest that EDRF(NO) may act as an endogenous free radical scavenger. This hypothesis was tested in an in vitro system by analyzing the effect of authentic NO (dilutions of a saturated aqueous solution) on .O2- production (detected spectrophotometrically as reduction of cytochrome c) by fMet-Leu-Phe-activated human leukocytes (PMN). NO depressed the rate of reduction of cytochrome c by .O2- released from PMN's or generated from the oxidation of hypoxanthine by xanthine oxidase. This effect was concentration-dependent and occurred at dilutions of the saturated NO solution (1:250 to 1:10) which inhibited platelet aggregation. NO had no direct effect on cytochrome c or on xanthine oxidase. These observations indicate that NO(EDRF) can be regarded as a scavenger of superoxide anion and they suggest that EDRF(NO) may provide a chemical barrier to cytotoxic free radicals (.O2-).     

Induction of antioxidant stress proteins in vascular endothelial and smooth muscle cells: protective action of vitamin C against atherogenic lipoproteins.

Elevated levels of lipid peroxidation and increased formation of reactive oxygen species within the vascular wall in atherosclerosis can overwhelm cellular antioxidant defence mechanisms. Accumulating evidence implicates oxidatively modified low density lipoproteins (LDL) in vascular dysfunction in atherosclerosis and oxidized LDL have been localized with in atherosclerotic lesions. We here report that human oxidatively modified LDL induce expression of 'antioxidant-like' stress proteins in vascular cells, involving increases in the activity of L-cystine transport, glutathione synthesis, heme oxygenase-1 and the murine stress protein MSP23. Moreover, treatment of human arterial smooth muscle cells with the dietary antioxidant vitamin C markedly attenuates adaptive increases in endogenous antioxidant gene expression and affords protection against smooth muscle cell apoptosis induced by moderately oxidized LDL. As vascular cell death is a key feature of atherosclerotic lesions and may contribute to the plaque 'necrotic' core, cap rupture and thrombosis, our findings suggest that the cytoprotective actions of vitamin C could limit plaque instability in advanced atherosclerosis.     

Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells

Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.     

氧化修饰低密度脂蛋白与动脉粥样硬化

动脉粥样硬化的发生发展与低密度脂蛋白受到氧化修饰有关。本文从以下四个方面对本室的工作进行了综述：（１）动脉粥样硬化机体受到脂质过氧化损伤;（２）Ｏｘ－ＬＤＬ对内皮细胞、平滑肌细胞和巨噬细胞的毒性效应;（３）Ｏｘ－ＬＤＬ和ＭＤＡ－ＬＤＬ的比较及与Ｏｘ－ＬＤＬ和ＭＤＡ－ＬＤＬ结合的清道夫受体的特征;（４）不同方法对ＬＤＬ氧化修饰的比较和以ＬＤＬ氧化修饰为模型对某些物质的抗氧化修饰研究。研究结果为动脉粥     

Oxidized low-density lipoprotein increases cultured human endothelial cell tissue factor activity and reduces protein C activation

Increasing evidence suggests that the formation of oxidized low-density lipoprotein (Ox-LDL) in vivo is associated with the development of atherosclerotic vascular disease. We investigated the effects of Ox-LDL on two vascular endothelial cell coagulant properties, tissue factor expression, and protein C activation. The Ox-LDL increased human arterial and venous endothelial cell tissue factor activity, with 100 micrograms/ml of Ox-LDL increasing factor activity fourfold. Native LDL modified by incubation with cultured human arterial and venous endothelial cells also induced endothelial cell tissue factor activity. This modification was blocked by coincubation with the antioxidants, probucol or ascorbic acid. It was determined, based on inhibition by known scavenger receptor antagonists (fucoidin, dextran sulfate), that binding of Ox-LDL via the acetyl LDL (scavenger) receptor was partially responsible for the increase in tissue factor expression. Whereas endothelial cell tissue factor expression was increased by incubation with Ox-LDL, protein C activation was reduced approximately 80% by incubating cultured endothelial cells with Ox-LDL. The effect of Ox-LDL on protein C activation was not inhibited by antagonists to the scavenger receptor. These data indicating that an atherogenic lipoprotein can regulate key vascular coagulant activities provide an additional link between vascular disease and thrombosis.     

Decomposition of lipid hydroperoxides enhances the uptake of low density lipoprotein by macrophages.

This study examined the roles of low-density lipoprotein (LDL) lipid oxidation and peroxide breakdown in its conversion to a form rapidly taken up by mouse peritoneal macrophages. Oxidation of the LDL without decomposition of the hydroperoxide groups was performed by exposure to gamma radiation in air-saturated solutions. Virtually complete decomposition of the hydroperoxides was achieved by treatment of the irradiated LDL with Cu2+ under strictly anaerobic conditions. No uncontrolled LDL uptake by macrophages occurred when the lipoprotein contained less than 150 hydroperoxide groups per particle. More extensively oxidized LDL was taken up and degraded by mouse macrophages significantly faster than the native lipoprotein. The uptake was greatly enhanced by treatment of the oxidized LDL with Cu2+. A significant proportion of the LDL containing intact or copper-decomposed LDL hydroperoxide groups accumulated within the macrophages without further degradation. Treatment of the radiation-oxidized LDL with Cu2+ was accompanied by aggregation of the particles. Competition studies showed that the oxidized LDL was taken up by macrophages via both the LDL and the scavenger receptors, whereas the copper-treated lipoprotein entered the cells only by the scavenger pathway. Phagocytosis also played an important role in the metabolism of all forms of the extensively modified LDL. Our results suggest that minimally-oxidized LDL is not recognized by the macrophage scavenger receptors unless the lipid hydroperoxide groups are decomposed to products able to derivatize the apo B protein.     

Low density lipoprotein receptor-related protein mediates apolipoprotein E inhibition of smooth muscle cell migration.

This research was undertaken to identify the cell surface receptor responsible for mediating apolipoprotein E (apoE) inhibition of platelet-derived growth factor (PDGF)-directed smooth muscle cell migration. Initial studies revealed the expression of the low density lipoprotein receptor (LDLR), the LDL receptor-related protein (LRP), the very low density lipoprotein receptor (VLDL), and apoE receptor-2 in mouse aortic smooth muscle cells. Smooth muscle cells isolated from LDLR-null, VLDL-null, and apoE receptor-2-null mice were responsive to apoE inhibition of PDGF-directed smooth muscle cell migration, suggesting that these receptors were not involved. An antisense RNA expression knockdown strategy, utilizing morpholino antisense RNA against LRP, was used to reduce LRP expression in smooth muscle cells to assess the role of this receptor in apoE inhibition of cell migration. Results showed that apoE was unable to inhibit PDGF-directed migration of LRP-deficient smooth muscle cells. The role of LRP in mediating apoE inhibition of PDGF-directed smooth muscle cell migration was confirmed by experiments showing that antibodies against LRP effectively suppressed apoE inhibition of PDGF-directed smooth muscle cell migration. Taken together, these results document that apoE binding to LRP is required for its inhibition of PDGF-directed smooth muscle cell migration.