Oxidized low density lipoprotein inhibits the migration of aortic endothelial cells in vitro.

Endothelial cell (EC) migration is a critical and initiating event in the formation of new blood vessels and in the repair of injured vessels. Compelling evidence suggests that oxidized low density lipoprotein (LDL) is present in atherosclerotic lesions, but its role in lesion formation has not been defined. We have examined the role of oxidized LDL in regulating the wound-healing response of vascular EC in vitro. Confluent cultures of bovine aortic EC were "wounded" with a razor, and migration was measured after 18 to 24 h as the number of cells moving into the wounded area and the mean distance of cells from the wound edge. Oxidized LDL markedly reduced migration in a concentration- and oxidation-dependent manner. Native LDL or oxidized LDL with a thiobarbituric acid (TBA) reactivity < 5 nmol malondialdehyde equivalents/mg cholesterol was not inhibitory; however, oxidized LDL with a TBA reactivity of 8-12 inhibited migration by 75-100%. Inhibition was half-maximal at 250-300 micrograms cholesterol/ml and nearly complete at 350-400 micrograms/ml. The antimigratory activity was not due to cell death since it was completely reversed 16 h after removal of the lipoprotein. The inhibitor molecule was shown to be a lipid; organic solvent extracts of oxidized LDL inhibited migration to nearly the same extent as the intact particle. When LDL was variably oxidized by dialysis against FeSO4 or CuSO4, or by UV irradiation, the inhibitory activity correlated with TBA reactivity and total lipid peroxides, but not with electrophoretic mobility or fluorescence (360 ex/430 em). This indicates that a lipid hydroperoxide may be the active species. These results suggest the possibility that oxidized LDL may limit the healing response of the endothelium after injury.

Fish oils inhibit endothelial cell production of platelet-derived growth factor-like protein.

Diets rich in fish and fish oils are associated with a reduced risk of cardiovascular disease and atherosclerosis. The interaction of a commercial fish oil extract (MaxEPA) with vascular endothelial cells (ECs) was studied as a possible mechanism for this protective effect. MaxEPA almost completely inhibited EC production of platelet-derived growth factor-like protein (PDGFc) while other lipids had a lesser effect or no effect. Overall protein synthesis was not reduced, nor was the inhibition due to defective secretion or increased degradation of the growth factor. Antioxidants suppressed the inhibitory activity of MaxEPA indicating that free radical oxidative processes were required for the inhibition. These results suggest that fish oils may suppress intimal smooth muscle cell proliferation by decreasing the production of EC-derived paracrine growth factors. This inhibitory process represents a possible molecular mechanism for the antiatherosclerotic action of marine lipids.

Role of ceruloplasmin in cellular iron uptake.

Individuals with hereditary ceruloplasmin (Cp) deficiency have profound iron accumulation in most tissues, which suggests that Cp is important for normal release of cellular iron. Here, in contrast to expectations, Cp was shown to increase iron uptake by HepG2 cells, increasing the apparent affinity for the substrate by three times. Consistent with its role in iron uptake, Cp synthesis was regulated by iron supply and was increased four- to fivefold after iron depletion. Unlike other iron controllers that are posttranscriptionally regulated, Cp synthesis was transcriptionally regulated. Thus, iron-deficient cells could increase Cp synthesis to maintain intracellular iron homeostasis, so that defects would lead to global accumulation of iron in tissues.

The surface of Mars: the view from the viking 2 lander.

Viking 2 lander began imaging the surface of Mars at Utopia Planitia on 3 September 1976. The surface is a boulder-strewn reddish desert cut by troughs that probably form a polygonal network. A plateau can be seen to the east of the spacecraft, which for the most probable lander location is approximately the direction of a tongue of ejecta from the crater Mie. Boulders at the lander 2 site are generally more vesicular than those near lander i. Fines at both lander sites appear to be very fine-grained and to be bound in a duricrust. The pinkish color of the sky, similar to that observed at the lander I site, indicates suspension of surface material. However, the atmospheric optical depth is less than that at the lander I site. After dissipation of a cloud of dust stirred during landing, no changes other than those stemming from sampling activities have been detected in the landscape. No signs of large organisms are apparent at either landing site.

Role of endogenous ceruloplasmin in low density lipoprotein oxidation by human U937 monocytic cells.

Oxidation of lipids and lipoproteins by macrophages is an important event during atherogenesis. Activation of monocytic cells by zymosan and other agonists results in the release of multiple oxidant species and consequent oxidation of LDL. We now show evidence that ceruloplasmin, a copper-containing acute phase reactant, is secreted by zymosan-activated U937 monocytic cells, and that the protein has an important role in LDL oxidation by these cells. In one approach, ceruloplasmin has been shown to exhibit oxidant activity under the appropriate conditions. Exogenous addition of purified human ceruloplasmin stimulates U937 cell oxidation of LDL to nearly the same extent as activation by zymosan. In contrast to previous cell-free experiments (Ehrenwald, E., G.M. Chisom, and P.L. Fox. 1994. Intact human ceruloplasmin oxidatively modifies low density lipoprotein. J. Clin. Invest. 93:1493-1501.) in which ceruloplasmin by itself (in PBS) oxidizes LDL, under the conditions of the current experiments (in RPMI 1640 medium) ceruloplasmin only oxidizes LDL in the presence of cells; the mechanism by which cells overcome the inhibition by medium components has not been ascertained. As further evidence for a role of ceruloplasmin, activation of U937 cells with zymosan induces ceruloplasmin mRNA and ceruloplasmin protein synthesis after a 5-6 h lag that is consistent with that preceding LDL oxidation. Finally, neutralization by a highly specific polyclonal antibody to human ceruloplasmin inhibits LDL oxidation by at least 65%. Moreover, multiple antisense oligodeoxynucleotides targeted to different regions of the ceruloplasmin mRNA block LDL oxidation by up to 95%. The specific action of the antisense oligonucleotides has been verified by showing inhibition of ceruloplasmin synthesis and by the ability of exogenous ceruloplasmin to overcome the inhibition. In summary, these results are consistent with a mechanism in which cell-derived ceruloplasmin participates in oxidation of LDL by U937 monocytic cells. The data also show that cellular factors in addition to ceruloplasmin, possibly active oxygen species and/or lipoxygenases, are essential and act synergistically with ceruloplasmin to oxidize LDL.

Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein.

Endothelial cell (EC) movement is required for the development and repair of blood vessels. We have previously shown that LDL oxidized by transition metals almost completely suppressed the wound-healing migratory response of vascular EC in vitro. We now report that lysophosphatidylcholine (lysoPC), a lipid component of oxidized LDL, has an important role in the antimigratory activity of the lipoprotein. Purified 1-palmitoyl lysoPC inhibited movement with a half-maximal activity at 12-15 micrometers, and near complete inhibition at 20 micrometers; the inhibitory concentration of lysoPC was consistent with its abundance in oxidized LDL. The inhibition was not due to cytotoxicity since protein synthesis was unaffected and since EC movement was restored after removal of lysoPC. Lysophospholipid activity was dependent on lipid structure. LysoPC's containing 1-position C16 or C18 saturated fatty acids were antimigratory, but those containing C < or = 14 saturated fatty acids or polyunsaturated fatty acids were not. The activity of 1-palmitoyl lysolipids with various head groups was examined. Lysophosphatidylinositol was more antimigratory than lysophosphatidylglycerol and lysophosphatidylcholine, which were more potent than lysophosphatidylserine and lysophosphatidylethanolamine. Monoglyceride was inactive while lysophosphatidate had promigratory activity. These results are consistent with head group size rather than charge as a critical determinant of activity. To show that lysophospholipids within an intact lipoprotein were active, LDL was treated with bee venom phospholipase A2 (PLA2). The modified lipoprotein inhibited EC movement to the same extent as iron-oxidized LDL and antimigratory activity correlated with the amount of lysoPC formed. To determine antimigratory activity of lysoPC present in oxidized LDL, lipid extracts from oxidized LDL were fractionated by normal phase HPLC. The fraction comigrating with lysoPC had nearly the same activity as the total extract confirming that lysoPC (or a co-eluting lipid) was a major antimigratory molecule in oxidized LDL. These studies demonstrate that lysoPC in oxidized LDL limit EC wound healing responses in vitro, and suggest a possible role for lysolipids in limiting endothelial regeneration after a denuding injury in vivo.

Intact human ceruloplasmin oxidatively modifies low density lipoprotein.

Ceruloplasmin is a plasma protein that carries most of the copper found in the blood. Although its elevation after inflammation and trauma has led to its classification as an acute phase protein, its physiological role is uncertain. A frequently reported activity of ceruloplasmin is its ability to suppress oxidation of lipids. In light of the intense recent interest in the oxidation of plasma LDL, we investigated the effects of ceruloplasmin on the oxidation of this lipoprotein. In contrast to our expectations, highly purified, undegraded human ceruloplasmin enhanced rather than suppressed copper ion-mediated oxidation of LDL. Ceruloplasmin increased the oxidative modification of LDL as measured by thiobarbituric acid-reacting substances by at least 25-fold in 20 h, and increased electrophoretic mobility, conjugated dienes, and total lipid peroxides. In contrast, ceruloplasmin that was degraded to a complex containing 115- and 19-kD fragments inhibited cupric ion oxidation of LDL, as did commercial preparations, which were also degraded. However, the antioxidant capability of degraded ceruloplasmin in this system was similar to that of other proteins, including albumin. The copper in ceruloplasmin responsible for oxidant activity was not removed by ultrafiltration, indicating a tight association. Treatment of ceruloplasmin with Chelex-100 removed one of seven copper atoms per molecule and completely blocked oxidant activity. Restoration of the copper to ceruloplasmin also restored oxidant activity. These data indicate that ceruloplasmin, depending on the integrity of its structure and its bound copper, can exert a potent oxidant rather than antioxidant action on LDL. Our results invite speculation that ceruloplasmin may be in part responsible for oxidation of LDL in blood or in the arterial wall and may thus have a physiological role that is quite distinct from what is commonly believed.

Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region.

Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.

Translational silencing of ceruloplasmin requires the essential elements of mRNA circularization: poly(A) tail, poly(A)-binding protein, and eukaryotic translation initiation factor 4G.

Ceruloplasmin (Cp) is a glycoprotein secreted by the liver and monocytic cells and probably plays roles in inflammation and iron metabolism. We showed previously that gamma interferon (IFN-gamma) induced Cp synthesis by human U937 monocytic cells but that the synthesis was subsequently halted by a transcript-specific translational silencing mechanism involving the binding of a cytosolic factor(s) to the Cp mRNA 3' untranslated region (UTR). To investigate how protein interactions at the Cp 3'-UTR inhibit translation initiation at the distant 5' end, we considered the "closed-loop" model of mRNA translation. In this model, the transcript termini are brought together by interactions of poly(A)-binding protein (PABP) with both the poly(A) tail and initiation factor eIF4G. The effect of these elements on Cp translational control was tested using chimeric reporter transcripts in rabbit reticulocyte lysates. The requirement for poly(A) was shown since the cytosolic inhibitor from IFN-gamma-treated cells minimally inhibited the translation of a luciferase reporter upstream of the Cp 3'-UTR but almost completely blocked the translation of a transcript containing a poly(A) tail. Likewise, a requirement for poly(A) was shown for silencing of endogenous Cp mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of PABP with the poly(A) tail or with eIF4G. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of PABP followed by quantitation of the poly(A) tail by reverse transcription-PCR and of eIF4G by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When PABP was depleted from the reticulocyte lysate with anti-human PABP antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for PABP. Similarly, in lysates treated with anti-human eIF4G antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for eIF4G. These data show that translational silencing of Cp requires interactions of three essential elements of mRNA circularization, poly(A), PABP, and eIF4G. We suggest that Cp mRNA circularization brings the cytosolic Cp 3'-UTR-binding factor into the proximity of the translation initiation site, where it silences translation by an undetermined mechanism. These results suggest that in addition to its important function in increasing the efficiency of translation, transcript circularization may serve as an essential structural determinant for transcript-specific translational control.

The regulation of endothelial cell motility by p21 ras.

Directed endothelial cell (EC) movement is required for the development and repair of blood vessels and plays a critical role in angiogenic processes obligatory for large tumor formation. We now report that ras proteins have a critical role in regulation of movement of normal mammalian cells. Bovine aortic EC microinjected with oncogenic Ha-ras enter further into an artificial wound than uninjected cells. Treatment with oncogenic Ha-ras also converts the cell paths from nearly linear in control cells to apparent 'random-walk' trajectories in treated cells, suggesting that oncogenic ras alters the normal control processes regulating cell motility. Botulinum toxin C blocks ras-stimulated motility indicating that a member of the p21 rho family is a downstream participant in the motile pathway. In related experiments we have observed that microinjection of the neutralizing, ras-specific, Y13-259 monoclonal antibody completely blocks both basal and basic fibroblast growth factor-stimulated movement of aortic EC. Y13-259 blocks the initiation of EC movement, as well as the continued progress of cells already in motion, suggesting that ras activity is continuously required throughout the motile process. Together these data indicate that ras is an integral component of the signaling pathway regulating cell movement.

High de novo synthesis of glycerolipids compared to deacylation-reacylation in rat liver microsomes.

A microsomal system characterized by high flux through the entire de novo pathway from glycerol phosphate to phosphatidylcholine and triacylglycerol has been developed. Optimum synthesis of phosphatidylcholine requires CDPcholine, Mg2+, KCl and a palmitoyl-CoA-generating system containing palmitic acid, ATP and CoA. Incorporation of [14C]glycerol phosphate into phosphatidylcholine/triacylglycerol synthesis ratio decreases as palmitate is increased. Phosphatidylcholine synthesis from glycerol phosphate is stimulated more by palmitate than by other saturated fatty acids; phosphatidylcholine synthesis increases with increasing unsaturation of the added fatty acids. The ratio of incorporation of [3H]palmitate to [14C]glycerol phosphate was determined for phosphatidic acid, diacylglycerol, phosphatidylcholine and triacylglycerol. This ratio is approximately 2 for all diacylglycerolipids and 3 for triacylglycerol. In our system, incorporation of palmitate into microsomal glycerolipid proceeds primarily by the de novo pathway, with minimal fatty acid recycling via deacylation-reacylation.

Ceruloplasmin and cardiovascular disease.

Transition metal ion-mediated oxidation is a commonly used model system for studies of the chemical, structural, and functional modifications of low-density lipoprotein (LDL). The physiological relevance of studies using free metal ions is unclear and has led to an exploration of free metal ion-independent mechanisms of oxidation. We and others have investigated the role of human ceruloplasmin (Cp) in oxidative processes because it the principal copper-containing protein in serum. There is an abundance of epidemiological data that suggests that serum Cp may be an important risk factor predicting myocardial infarction and cardiovascular disease. Biochemical studies have shown that Cp is a potent catalyst of LDL oxidation in vitro. The pro-oxidant activity of Cp requires an intact structure, and a single copper atom at the surface of the protein, near His(426), is required for LDL oxidation. Under conditions where inhibitory protein (such as albumin) is present, LDL oxidation by Cp is optimal in the presence of superoxide, which reduces the surface copper atom of Cp. Cultured vascular endothelial and smooth muscle cells also oxidize LDL in the presence of Cp. Superoxide release by these cells is a critical factor regulating the rate of oxidation. Cultured monocytic cells, when activated by zymosan, can oxidize LDL, but these cells are unique in their secretion of Cp. Inhibitor studies using Cp-specific antibodies and antisense oligonucleotides show that Cp is a major contributor to LDL oxidation by these cells. The role of Cp in lipoprotein oxidation and atherosclerotic lesion progression in vivo has not been directly assessed and is an important area for future studies.

Structure, oxidant activity, and cardiovascular mechanisms of human ceruloplasmin.

Ceruloplasmin is the principal carrier of copper in human plasma. It is an abundant protein that participates in the acute phase reaction to stress, but its physiological function(s) is unknown. An antioxidant activity of ceruloplasmin has been described, but recent evidence suggests that the protein may also exhibit potent pro-oxidant activity and cause oxidative modification of low density lipoprotein (LDL). The pro-oxidant activity is highly dependent on the structure of the protein; removal of a single one of the seven integral copper atoms, or a specific proteolytic cleavage event, completely suppresses LDL oxidation. This newly described pro-oxidant activity may help to explain epidemiological studies indicating that ceruloplasmin is an independent risk factor for cardiovascular disease.

Environmental modifications in the homes of elderly Canadians with disabilities.

This study was performed to (1) determine if the use of architectural modifications in the homes of the disabled elderly can reduce the level of disability; (2) determine the predictors of the use of architectural modifications; and (3) estimate the degree of need for environmental modifications. The degree of need was estimated using data from 8895 respondents (55 years and older) from the Canadian Health and Disability Survey (1983-84). A subgroup of 1520 respondents who needed architectural modifications was selected to predict: (1) the ability to perform instrumental activities of daily living (IADL) and (2) the use of architectural modifications. These data were analysed using multiple linear regression. Approximately 37.9% of the disabled elderly required at least one modification to their home. About 40% did not have the architectural modification that was needed. Income was found to be a predictor of the use of architectural modifications (p = 0.0273). Users of architectural modifications were more independent in IADL than non-users (p < 0.0312). In conclusion, there is a substantial need for environmental modifications in the homes of the disabled elderly. Furthermore, architectural modifications should be available at a low cost.

Ceruloplasmin copper induces oxidant damage by a redox process utilizing cell-derived superoxide as reductant.

Oxidative damage by transition metals bound to proteins may be an important pathogenic mechanism. Ceruloplasmin (Cp) is a Cu-containing plasma protein thought to be involved in oxidative modification of lipoproteins. We have previously shown that Cp increased cell-mediated low-density lipoprotein (LDL) oxidation by a process requiring cell-derived superoxide, but the underlying chemical mechanism(s) is (are) unknown. We now show that superoxide reduction of Cp Cu is a critical reaction in cellular LDL oxidation. By bathocuproine disulfonate (BCS) binding and by superoxide utilization, we showed that exogenous superoxide reduces a single Cp Cu atom, the same Cu required for LDL oxidation. The Cu atom remained bound to Cp during the redox cycle. Three avenues of evidence showed that vascular cells reduce Cp Cu by a superoxide-dependent process. The 2-fold higher rate of Cp Cu reduction by smooth muscle cells (SMC) compared to endothelial cells (EC) was consistent with their relative rates of superoxide release. Furthermore, Cp Cu reduction by cells was blocked by Cu,Zn superoxide dismutase (SOD1). Finally, the level of superoxide produced by EC and SMC was sufficient to cause the amount of Cu reduction observed. An important role of Cp Cu reduction in LDL oxidation was suggested by results showing that SOD1 inhibited Cp Cu reduction and LDL oxidation by SMC with equal potency, while tumor necrosis factor-alpha stimulated both processes. In summary, these results show that superoxide is a critical cellular reductant of divalent transition metals involved in oxidation, and that protein-bound Cu is a substrate for this reaction. The role of these mechanisms in oxidative processes in vivo has yet to be defined.

Regulation of production of a platelet-derived growth factor-like protein by cultured bovine aortic endothelial cells.

Platelet-derived growth factor (PDGF) is a potent mitogen for cultured cells of mesenchymal origin. Known sources of PDGF or PDGF-like protein are blood platelets, several transformed cell lines, and cultured endothelial cells (EC). We have examined the regulation of production of a PDGF-like protein in cultures of bovine aortic EC using a specific radioreceptor assay for PDGF. EC constitutively secreted PDGF-like protein into serum-containing or serum-free medium. The rate of production of PDGF-like protein was constant for at least 3 weeks and was not due to release of an internal store, since cell lysis by repeated freeze/thaw cycles did not release significant amounts of the protein. Synthesis of PDGF-like protein was sensitive to changes in the pH of the media and was maximal at pH 8.5. Production of PDGF-like protein was independent of EC growth rate: rapidly dividing cells and confluent, quiescent cells produced equal amounts per cell. However, sparse, quiescent EC produced more PDGF-like protein per cell than did confluent, quiescent cells. Several phorbol esters stimulated production of PDGF-like protein. At a concentration of 10(-6) M, a twofold stimulation was observed upon addition of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and nearly a fourfold stimulation upon addition of the nonpromoting analog, methyl TPA. Incubation of EC with endotoxin (10 micrograms/ml) resulted in a twofold stimulation of PDGF-like protein production. In all experiments with endotoxin and phorbol esters, an increase in the production of PDGF-like protein was accompanied by morphological changes in the EC cultures. The cells appeared elongated and fibroblastic and exhibited low viability. A mathematical model was developed in which PDGF-like protein production was shown to consist of two separate components--production at a constant rate by healthy cells and a large burst of synthesis and secretion by dying cells. These results suggest that injurious agents may be capable of stimulating production of a growth factor by the endothelium.

Modified low density lipoproteins suppress production of a platelet-derived growth factor-like protein by cultured endothelial cells.

Cultured endothelial cells (EC) produce a platelet-derived growth factor-like protein (PDGF-c) that stimulates the growth of cultured cells of mesenchymal origin. We have examined the effect of native plasma low density lipoprotein (LDL) and chemically modified LDL on production of PDGF-c by EC. Acetyl-LDL, but not native LDL, suppressed the production of PDGF-c by bovine aortic EC. Half-maximal inhibition was observed at a concentration of 25-75 micrograms of cholesterol per ml, and maximal inhibition (0-25% of control) at 150 micrograms of cholesterol per ml. EC treated with acetyl-LDL showed no morphological damage, there was no change in cell number, and the effect on production of PDGF-c was substantially reversed upon removal of the acetyl-LDL. The observed inhibition of PDGF-c production was specific, since total cellular and secreted protein synthesis were unaffected by acetyl-LDL. Acetyl-LDL suppressed PDGF-c production in both bovine aortic and human umbilical vein EC, but not in rat heart EC. This cell specificity correlated with the presence of scavenger receptors as measured by degradation of 125I-labeled acetyl-LDL and uptake of fluorescently labeled acetyl-LDL. Dimethylpropanediamine-LDL, a cationic modified lipoprotein, also inhibited PDGF-c production. The inhibition by both types of modified LDL was accompanied by significant intracellular cholesterol accumulation, suggesting a role for EC lipid composition in the regulation of production of PDGF-c.