Cellular Thiol Production and Oxidation of Low-Density Lipoprotein

Compelling evidence suggests that low-density lipoprotein (LDL) is oxidized by cells within the arterial intima and that, once oxidized, it is profoundly atherogenic. The precise mechanism(s) by which cells promote the oxidation of LDL in vivo are not known; in vitro, however, oxidation of LDL can be enhanced by a number of differing mechanisms, including reaction with free and protein-bound metal ions, thiols, reactive oxygen species, lipoxygenase, myeloperoxidase and peroxynitrite. This review is concerned with the mechanisms by which cells enhance the oxidation of LDL in the presence of transition metals; in particular, the regulation, pro- and anti-oxidant consequences, and mechanism of action of cellular thiol production are examined, and contrasted with thiol-independent oxidation of LDL in the presence of transition metals.     

Cell-mediated LDL oxidation: the impact of transition metals and transferrin

Activated monocytes release oxygen radicals by respiratory burst and oxidative damage can be accelerated by transition metals. We investigated the cell-mediated and metal-catalysed in vitro oxidation of low-density lipoproteins (LDL), as well as the impact of the metal-binding protein transferrin (Tf). LDL oxidation was measured by monitoring the increase in fluorescence (350/440 nm excitation/emission). Maximal respiratory burst by U937 cells was achieved after 96 h differentiation with retinoic acid and dihydroxyvitamin D3 followed by stimulation with opsonised zymosan. Addition of activated cells resulted in the LDL oxidation, even in the absence of transition metals. Moreover, activated cells greatly enhanced metal-catalysed oxidative modifications, especially in the presence of copper. By binding metals, Tf was able to strongly impair this process. In conclusion, by generating oxygen radicals, activated U937 cells were able to oxidise LDL. The oxidising process was most pronounced in the presence of copper and could be blocked by Tf.     

Oxidative modification of low density lipoprotein (LDL) by activated human monocytes and the cell lines U937 and HL60

Summary Human peripheral blood monocytes, upon activation, have the capacity to oxidize low density lipoprotein (LDL) and render the LDL toxic to cultured cells. Previous studies by our laboratory indicate that this process is mediated by free radicals in that it can be prevented by addition of free radical scavengers and antioxidants during the incubation of monocytes with LDL. Here we report that optimal modification of LDL by monocytes was influenced by media composition. In the absence of added metal ions, oxidation was distinctly dependent on the concentration of monocytes as well as LDL concentration. Exposure of monocytes to lipopolysaccharide or stimulation of phagocytosis by opsonized zymosan resulted in marked enhancement of LDL oxidation compared to other activating agents. After exposure to activated monocytes, lipid oxidation products in the supernatant were found both in a high molecular weight fraction containing LDL (>30 000 Daltons) and in a lipoprotein-free, low molecular weight fraction (<30 000 Daltons), yet only the high molecular weight, LDL-containing fraction was toxic to target cells. In addition, human myelomonocytic cell lines U937 and HL60 were shown to mediate oxidation of LDL. As with monocytes, exposing these cells to opsonized zymosan caused the level of LDL oxidation to be significantly enhanced. These findings offer further insight into the mechanisms of monocyte-mediated oxidation of lipoproteins and will facilitate studies investigating the role of monocyte-modified LDL in tissue injury. This project was funded by grants form the American Heart Association-Northeast Ohio Affiliate and the National Institutes of Health, Bethesda, MD (HL-29582).     

Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells.

A high concentration of circulating low-density lipoproteins (LDL) is a major risk factor for atherosclerosis. Native LDL and LDL modified by glycation and/or oxidation are increased in diabetic individuals. LDL directly stimulate vascular smooth muscle cell (VSMC) proliferation; however, the mechanisms remain undefined. The extracellular signal-regulated kinase (ERK) pathway mediates changes in cell function and growth. Therefore, we examined the cellular effects of native and modified LDL on ERK phosphorylation in VSMC. Addition of native, mildly modified (oxidized, glycated, glycoxidized) and highly modified (highly oxidized, highly glycoxidized) LDL at 25 microg/ml to rat VSMC for 5 min induced a fivefold increase in ERK phosphorylation. To elucidate the signal transduction pathway by which LDL phosphorylate ERK, we examined the roles of the Ca(2+)/calmodulin pathway, protein kinase C (PKC), src kinase, and mitogen-activated protein kinase kinase (MEK). Treatment of VSMC with the intracellular Ca(2+) chelator EGTA-AM (50 micromol/l) significantly increased ERK phosphorylation induced by native and mildly modified LDL, whereas chelation of extracellular Ca(2+) by EGTA (3 mmol/l) significantly reduced LDL-induced ERK phosphorylation. The calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (40 micromol/l) significantly decreased ERK phosphorylation induced by all types of LDL. Downregulation of PKC with phorbol myristate acetate (5 micromol/l) markedly reduced LDL-induced ERK phosphorylation. Pretreatment of VSMC with a cell-permeable MEK inhibitor (PD-98059, 40 micromol/l) significantly decreased ERK phosphorylation in response to native and modified LDL. These findings indicate that native and mildly and highly modified LDL utilize similar signaling pathways to phosphorylate ERK and implicate a role for Ca(2+)/calmodulin, PKC, and MEK. These results suggest a potential link between modified LDL, vascular function, and the development of atherosclerosis in diabetes.     

Low-density lipoprotein modification by normal,myeloperoxidase-deficient and NADPH oxidase-deficient granulocytes and the impact of redox active transition metal ions

The modification of low-density lipoprotein (LDL) by normal, myeloperoxidase (MPO)-deficient and NADPH oxidase-deficient granulocytes was investigated using the monoclonal antibody (mAb) OB/04, which was originally generated against copper-oxidized LDL. Incubation of LDL with normal granulocytes increased the reactivity of LDL with mAb OB/04. These effects were even more pronounced using MPO-deficient granulocytes. Inhibitors of oxidative reactions (the NADPH oxidase inhibitor diphenyleneiodonium chloride [DPI], catalase, superoxide dismutase [SOD]) did not significantly reduce LDL oxidation by normal granulocytes. Furthermore, granulocytes of a patient with NADPH oxidase deficiency were almost equally effective as normal granulocytes, indicating that oxidative burst-derived reactive oxygen species are of only minor importance in the generation of mAb OB/04-detectable new epitopes on LDL in vitro. In contrast, incubation of LDL with iron and copper prior to and during incubation with normal granulocytes markedly enhanced the generation of OB/04-detectable epitopes. It is supposed that, besides superoxide (in normal and MPO-deficient granulocytes) or instead of superoxide (in NADPH oxidase-deficient granulocytes), lytic enzymes released by activated granulocytes may enhance the availability of transition metals for oxidation of LDL. Our results support the concept that transition-metal-dependent pathways of LDL oxidation in combination with degranulation products of granulocytes are important.     

HIV-1 Tat induces TNF-alpha production by human monocytes: involvement of calcium and PKC pathways

In this study we investigated the signaling pathways triggered by Tat in human monocyte to induce TNF-alpha. In monocytes, calcium, PKA, and PKC pathways are highly implicated in the expression of cytokine genes. Our data show that (i) extracellular calcium is required for the calcium signal initiated by Tat in the monocyte and is required for TNF-alpha production, PKC pathway is also required, whereas the PKA pathway does not seem to be involved (ii) downstream from PKC, activation of NF kappa B is essential while ERK1/2 MAP kinases, even though activated by Tat, are not directly involved in the pathway signaling leading to TNF-alpha production.     

Activation of protein kinase C by phorbol esters in human macrophages reduces the metabolism of modified LDL by down-regulation of scavenger receptor activity

Atherogenesis and inflammation are dependent on macrophage function. Signalling pathways are involved in the modulation of the classical low density lipopotein (LDL)-receptor and scavenger receptors activities, which are both expressed by macrophages. This study has evaluated the role of activation of the protein kinase A and C pathways in human macrophages on the metabolism of lipid carried by native, acetylated and oxidised LDL. We found that [3H]oleate incorporation into cholesteryl ester and triacylglycerol is increased by an analogue of cAMP, but strongly inhibited by treatment with phorbol ester (PMA) (100 nM, 6 h) in the presence of acLDL and oxLDL and, to a lesser extent, nLDL. The mechanisms underlying the effects of the phorbol ester were investigated further. The protein kinase C inhibitors, calphostin C and herbimycin A, prevented the PMA-mediated inhibition of cholesterol esterification. PMA also reduced [14C]acetate incorporation into newly synthesised lipids especially in the presence of nLDL, and reduced the uptake of cholesterol carried by modified LDL. Furthermore, the effects of PMA were not modified by inhibition of proteases activities, ruling out the hypothesis that CD163, a scavenger receptor which is shed by the cell surface in the presence of phorbol, is involved in the phorbol-induced reduction of cholesterol accumulation in macrophages in response to LDL. We conclude that binding of modified LDL to macrophages induces an appropriate pattern of scavenger receptor phosphorylation which, in turn, determines the optimal receptor internalisation process. PMA activates PKC pathways and prevents the optimal ligand-induced phosphorylation of the receptors, compromising the processes of degradation of modified LDL. The data also suggest that this mechanism may be related to the decreased uptake by activated macrophages of lipid carried by modified lipoproteins during the early phases of inflammation (284).     

Oxidation of low-density lipoprotein by thiol compounds leads to its recognition by the acetyl LDL receptor

Reduced glutathione and other compounds with free -SH groups promoted the oxidation of low-density lipoprotein (LDL) in the absence of cells in Ham's F-10 medium. In contrast, compounds in which the thiol groups were oxidized or blocked were ineffective in oxidizing LDL. Thiol-induced modification of LDL did not occur in media lacking in redox metals. It is suggested that thiols react with redox metal, generating thiol- and oxygen-derived free radicals that promote modification of LDL.     

Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O2*-) production by peripheral blood monocytes. We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O2*- production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O2*- production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O2*- than those from controls. Nifedipine treatment considerably decreased O2*- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O2*- production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O2*- overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress.     

Redistribution of metal ions to control low density lipoprotein oxidation in Ham's F10 medium

The study of cell-mediated low density lipoprotein (LDL) oxidation has traditionally been undertaken using Ham's F10 media due to its high metal content and low levels of antioxidants. Although there has been no acknowledged change to this media in recent years by the suppliers, Ham's F10 medium has been found to be extremely inconsistent in its promotion of LDL oxidation in the absence of cells. This variability contrasts with the relatively consistent rates of THP-1 cell-mediated LDL oxidation. This study has now shown that the variability in cell-free LDL oxidation is medium-dependent and not an artefact of experimental protocol. It presents evidence that suggests the variable rates of cell-free LDL oxidation are caused by iron auto-oxidation during storage of the Ham's F10 medium. The medium can be standardized by removal of all transition metals, by treatment with Chelex, before the addition of known amounts of iron or copper. This treatment generates a cell culture medium that only allows very slow LDL oxidation in the absence of cells.     

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.     

Protein kinase C beta is required for human monocyte chemotaxis to MCP-1

Monocyte chemoattractant protein 1 (MCP-1) is important in attracting monocytes to sites of inflammation. Using predominantly pharmacological approaches, prior studies have indicated that serine/threonine kinases are involved in the MCP-1-induced signaling pathways. We report here that there is substantial inhibition of MCP-1-stimulated chemotaxis of human monocytes treated with inhibitors selective for the subset of serine/threonine kinases, protein kinase C (PKC). Selective inhibitors of PKC such as GF109203X and Calphostin C both caused approximately 80% inhibition of chemotaxis. Because these pharmacological inhibitors do not specifically inhibit individual PKC isoforms, we chose to use antisense oligodeoxyribonucleotides (ODN) to specifically reduce PKC isoform expression, first by inhibiting expression of the conventional PKC family, and next by using specific antisense ODN for PKCalpha and PKCbeta. Conventional PKC-antisense ODN treatment completely and significantly inhibited monocyte chemotaxis to MCP-1, whereas sense-control ODN caused no significant inhibition. PKCbeta-antisense ODN caused 89.2% inhibition of chemotaxis at its highest dose. In contrast, PKCbeta-sense ODN and PKCalpha-antisense and -sense ODN were without effect. Further studies evaluating the calcium response that is triggered upon MCP-1 interaction with its receptor, CCR2, indicate that this response is not altered by antisense or sense ODN treatment, thus supporting our hypothesis that PKCbeta is critical for post-receptor signal transduction downstream of the immediate calcium signal. These data contribute to our developing understanding of the signal transduction pathways involved in the chemotactic response of human monocytes to MCP-1 and uniquely identify the requirement for the PKCbeta isoform in this important process.     

An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu²⁺, Ni²⁺, Co²⁺, and Zn²⁺). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.     

蛋白激酶C亚型在HL—60细胞诱导分化中的变化

用全反式维甲酸（ＡＴＲＡ）或佛波酯（ＰＭＡ）处理人早幼粒白血病细胞（ＨＬ－６０）３天,用形态学,ＮＢＴ还原实验,特异性和非特异性酯酶测定,证明细胞分别向粒细胞或单核／巨噬细胞分化。通过免疫组化法观察了蛋白激酶Ｃ（ＰＫＣ）α,βⅠ和βⅡ亚型在分化后的变化。结果显示,ＡＴＲＡ可引起ＨＬ－６０细胞ＰＫＣα,βⅠ和βⅡ的含量升高,分别为对照的５．０,２．８和４．２倍,并存在从胞膜向胞质转位。ＰＭＡ则使ＰＣ     

Crosstalk of sterol-dependent and non-sterol-dependent signaling in human monocytes after in vitro addition of LDL

The aim of the present study was to investigate low density lipoprotein (LDL)-induced, non-sterol-dependent signaling and its possible role in cholesterol balance. LDL in 10 microg ml(-1) concentration could induce inositol trisphosphate (IP3) and Ca2+ signal generation through a pertussis toxin (PT) sensitive G protein in human monocytes. The increase in [Ca2+]i was derived from the intracellular pools. LDL also induced activation and translocation of protein kinase C (PKC) into the cell membrane, by processes, which were significantly inhibited in the first 20 min by preincubation with PT and PKC-inhibitor H-7. The PKC-activating phorbol-12-myristate-13-acetate (PMA), differently from LDL, enhanced the LDL-receptor (LDL-R)-mediated binding and degradation of [125I]LDL, but inhibited endogenous cholesterol synthesis, and both effects were inhibited by H-7. The LDL-induced inhibition of binding and degradation of [125I]LDL was not affected by H-7, whereas decreased cholesterol synthesis was counteracted by H-7. These results suggest the existence of a non-sterol-dependent signal pathway of LDL-Rs, by which endogenous cholesterol synthesis, that is, the [14C]acetate incorporation, is regulated through PKC activation.     

Glucose enhancement of LDL oxidation is strictly metal ion dependent

Recent evidence suggests that lipoprotein oxidation is increased in diabetes, however, the mechanism(s) for such observations are not clear. We examined the effect of glucose on low-density lipoprotein (LDL) oxidation using metal ion-dependent and -independent oxidation systems. Pathophysiological concentrations of glucose (25 mM) enhanced copper-induced LDL oxidation as determined by conjugated diene formation or relative electrophoretic mobility (REM) on agarose gels. Similarly, iron-induced LDL oxidation was stimulated by glucose resulting in 4- to 6-fold greater REM than control incubations without glucose. In contrast, glucose had no effect on metal ion-independent LDL oxidation by aqueous peroxyl radicals. The effect of glucose on metal ion-dependent LDL oxidation was associated with enhanced reduction of metal ions, and in the case of iron-induced LDL oxidation, was completely inhibited by superoxide dismutase. The effect of glucose was mimicked by other reducing sugars, such as fructose and mannose, and the extent to which each sugar enhanced LDL oxidation was closely linked to its metal ion-reducing activity. Thus, promotion of LDL oxidation by glucose is specific for metal ion-dependent oxidation and involves increased metal ion reduction. These results provide one potential mechanism for enhanced LDL oxidation in diabetes.