Elliott Sigal

Gene expression in macrophage-rich human atherosclerotic lesions. 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts.

Oxidatively modified low density lipoprotein (LDL) exhibits several potentially atherogenic properties, and inhibition of LDL oxidation in rabbits decreases the rate of the development of atherosclerotic lesions. In vitro studies have suggested that cellular lipoxygenases may be involved in LDL oxidation, and we have shown previously that 15-lipoxygenase and oxidized LDL are present in rabbit atherosclerotic lesions. We now report that epitopes of oxidized LDL are also found in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions. Using in situ hybridization and immunostaining techniques, we also report that 15-lipoxygenase mRNA and protein colocalize to the same macrophage-rich areas. Moreover, these same lesions express abundant mRNA for the acetyl LDL receptor but no detectable mRNA for the LDL receptor. We suggest that atherogenesis in human arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase, leading to subsequent enhanced macrophage uptake, partly by way of the acetyl LDL receptor.

Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system.

Human prostaglandin G/H synthase 1 and 2 were expressed in the baculovirus expression system and purified to high levels. Both enzymes were glycosylated. PGHS-1 appeared to be homogeneous by SDS-PAGE analysis but two closely migrating bands were detected in PGHS-2 preparation which were evidently due to heterogeneity in glycosylation. The amino-acid sequence of the N-termini of both isoforms indicated that the signal sequences were efficiently cleaved by the insect cells. The recombinant human PGHS-1 and PGHS-2 possessed both cyclooxygenase and peroxidase activities. Both had high affinities for arachidonate as substrate and underwent self-inactivation during catalysis. The recombinant isoforms were not pharmacologically identical, since some NSAIDs were selective inhibitors of either PGHS-1 or PGHS-2. This is the first report of high levels of expression and purification of human PGHS isoforms. The recombinant enzymes are invaluable in developing potent PGHS-2 selective inhibitors that may be efficacious anti-inflammatory drugs with no or low levels of toxicity.

Overexpression of 15-Lipoxygenase in Vascular Endothelium Accelerates Early Atherosclerosis in LDL Receptor–Deficient Mice

To study the possible role of the human lipid-oxidizing enzyme 15-lipoxygenase (15-LO) in atherosclerosis, we overexpressed it specifically in the vascular wall of C57B6/SJL mice by using the murine preproendothelin-1 promoter. The mice overexpressing 15-LO were crossbred with low density lipoprotein (LDL) receptor–deficient mice to investigate atherogenesis. High levels of 15-LO were expressed in the atherosclerotic lesion in the double-transgenic mice as assessed by immunohistochemistry. The double-transgenic, 15-LO–overexpressing, LDL receptor–deficient mice (LDLR−/−/15LO) developed significantly larger atherosclerotic lesions at the aortic sinus compared with lesions in the LDL receptor–deficient (LDLR−/−) mice after 3 and 6 weeks (107 000 versus 28 000 &mgr;m2 [P <0.001] and 121 000 versus 87 000 &mgr;m2 [P <0.05], respectively) of an atherogenic diet. LDL from the LDLR−/−/15LO mice was more susceptible to oxidation than was the LDL from the control LDLR−/− mice, as shown by a shorter lag period for copper-induced conjugated diene formation. On the other hand, no differences were found in the levels of serum anti–oxidized LDL antibodies between the study groups. There were also no differences with respect to the density of macrophages and T lymphocytes infiltrating the lesions in both experimental groups. Taken together, these results support the hypothesis that 15-LO overexpression in the vessel wall is associated with enhanced atherogenesis.

Molecular cloning and primary structure of human 15-lipoxygenase

A full-length cDNA encoding 15-lipoxygenase has been isolated from a human reticulocyte cDNA library. The predicted primary structure of the enzyme exhibits a sequence similarity of 61% and 45% with human 5-lipoxygenase and the soybean lipoxygenase isoenzyme I, respectively. When all three lipoxygenases are aligned, there are two distinct regions of significant sequence identity including a cluster of five histidine residues conserved in all three lipoxygenases. Because histidines can serve as ligands for the enzymatically active iron, this region may be critical to enzymatic function. These results provide a basis for exploring functional domains of lipoxygenases.

Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells.

In reticulocytes, the enzyme 15-lipoxygenase (15-LO) is believed to contribute to cellular differentiation, and in leukocytes and airway cells 15-LO generates inflammatory mediators. The recent availability of antibodies to 15-LO now allows us to determine which specific cells contain the enzyme, to characterize its subcellular localization, and to determine its expression at the translational level. A polyclonal antibody to recombinant human reticulocyte 15-LO was used with a standard immunofluorescent technique. In rabbit red blood cells, fluorescence appeared during the course of anemia. Early reticulocytes did not fluoresce, but more mature reticulocytes showed increased fluorescent intensity. Late reticulocytes contained little fluorescence. Among human leukocytes, only eosinophils fluoresced. In human trachea, 15-LO immunofluorescence was localized to epithelial cells, and both basal and ciliated cells fluoresced. In all cells studied, fluorescence was localized to the cytoplasm and was variable in degree among cells in each preparation. We conclude that the 15-LO of airway cells and eosinophils is immunologically related to the reticulocyte 15-LO. Furthermore, the variable fluorescence among cells (e.g., in epithelium) and during development (e.g., reticulocytes) suggests a role of 15-LO in cell growth and development.

Overexpression, purification and characterization of human recombinant 15-lipoxygenase

Human 15-lipoxygenase was expressed to high levels (approx. 20% of cellular protein) in a baculovirus/insect cell expression system. Catalytically active enzyme was readily purified (90-95% pure) from cytosolic fractions by anion-exchange chromatography on a Mono Q column with approx. 95% recovery of enzymatic activity. Routinely, a yield of 25-50 mg of pure enzyme per L of culture and a specific activity of 7.1-21 mumol 13-hydroxyoctadecadienoic acid (13-HODE)/mg.min (turnover rate of 8.4-25 s-1) were obtained. Both the specific activity and the enzymes iron content was significantly increased by the addition of ferrous ions to either the purified enzyme or to the insect cell culture medium during production. An isoelectric point of 5.85 was determined and the N-terminal amino acid sequence was found to be identical to that predicted from the cDNA. The purified recombinant enzyme exhibits a dual positional specificity with arachidonic acid (formation of 15S- and 12S-hydroxyeicosatetraenoic acid (12S-HETE) in a ratio of 12:1). Double oxygenation products 14R,15S- and various 8,15-DiHETE isomers were also identified. With linoleic acid as substrate, a pH-optimum of 7.0 and a KM of 3 microM were determined. The enzyme undergoes suicidal inactivation during fatty acid oxygenation, is sensitive to standard lipoxygenase inhibitors, and oxygenates phospholipids, cholesterol esters, biomembranes and human low-density lipoprotein. Contrary to prior studies on the rabbit enzyme, no glycosylation was detected.

DEFINING THE ARACHIDONIC ACID BINDING SITE OF HUMAN 15-LIPOXYGENASE : MOLECULAR MODELING AND MUTAGENESIS

Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory disorders and are, therefore, important targets for drug discovery. Both plant and mammalian lipoxygenases catalyze the dioxygenation of polyunsaturated fatty acids, which contain one or more 1,4-cis,cis-pentadiene units to yield hydroperoxide products. At the time this study was initiated, soybean lipoxygenase-1 was the only lipoxygenase for which an atomic resolution structure had been determined. No structure of lipoxygenase with substrate or inhibitor bound is currently available. A model of arachidonic acid docked into the proposed substrate binding site in the soybean structure is presented here. Analysis of this model suggested two residues, an aromatic residue and a positively charged residue, could be critical for substrate binding. Validation of this model is provided by site-directed mutagenesis of human 15-lipoxygenase, despite the low amino acid sequence identity between the soybean and mammalian enzymes. Both a positively charged amino acid residue (Arg402) and an aromatic amino acid residue (Phe414) are identified as critical for the binding of fatty acid substrates in human 15-lipoxygenase. Thus, binding determinants shown to be characteristic of non-enzymatic fatty acid-binding proteins are now implicated in the substrate binding pocket of lipoxygenases.

Arachidonate 15-lipoxygenase from human eosinophil-enriched leukocytes: Partial purification and properties

Arachidonate 15-lipoxygenase was purified from human eosinophil-enriched leukocytes after showing that 15-lipoxygenase activity was 100-fold greater in eosinophils than in neutrophils. Partial purification was achieved using ammonium sulfate precipitation, cation-exchange and hydrophobic-interaction chromatography. New evidence is presented suggesting that 15-lipoxygenase has electrostatic and hydrophobic properties distinct from 5-lipoxygenase. In addition, ATP is shown to inhibit, and phosphatidylcholine is shown to stimulate, 15-lipoxygenase, suggesting a regulatory role for these compounds in the lipoxygenation of arachidonic acid.

Cloning and characterization of a murine macrophage lipoxygenase

We have isolated a murine macrophage cDNA encoding a 12-lipoxygenase, that represents the homolog of the human 15-lipoxygenase. The predicted amino acid sequence of this lipoxygenase is highly similar to the rat 12-lipoxygenase isolated from brain and human 15-lipoxgenase. The recombinant enzyme expressed in Cos-7 cells oxidizes arachidonic acid to 12- and 15-HETE with a profile similar to that obtained from peritoneal macrophages. A polyclonal antibody generated against a putative peptide recognizes a 75 kDa protein in cell extracts from mouse peritoneal macrophages and transfected Cos-7 cells. The lipoxygenase cDNA hybridizes to a 2.5 kb mRNA present in peritoneal macrophages, lung, spleen, heart and liver. RT-PCR analysis indicates that the same lipoxygenase is expressed in mouse reticulocytes. A partial genomic clone for this lipoxygenase has also been characterized. Southern blot analysis of mouse genomic DNA indicates that this is a single copy gene.

Oxidation, Lipoxygenase, and Atherogenesis

Mounting evidence suggests that oxidative processes contribute to the pathogenesis of atherosclerosis and that antioxidants may represent a strategy to complement the lowering of lipids in the therapy of this disease. Although multiple molecular events have been identified in vitro and although it is tempting to ascribe multiple atherogenic properties to oxidized LDL, our understanding of this process remains incomplete. Further research is warranted in several areas. First, it will be important to selectively inhibit different aspects of the process to determine the relative contribution of various biological targets. In this regard pharmacological inhibition of 15-lipoxygenase in vivo in relevant animal models is required to address the question of the contribution of this enzyme to significant oxidative events. The lack of specific inhibitors has made this task more difficult. It will also be important to define the biologically active moiety of oxidized LDL to begin to determine the mechanisms through which it exerts its atherogenic effects. It is likely that alternate protein targets can be identified both downstream and upstream of the oxidative process. Research is only now beginning to elucidate the inflammatory mechanisms that account for the cellular response. Further research into adhesion events, cytokine profiles, and downstream effector molecules of the oxidative process are likely to identify alternate targets for therapeutic intervention.