Previous studies in this laboratory established that low density lipoprotein (LDL) incubated with cultured endothelial cells, smooth muscle cells, or macrophages undergoes a free radical-catalyzed oxidative modification that generates lipid peroxides and extensive structural changes in the LDL molecule. The oxidatively modified LDL strongly inhibited chemotactic responses of the mouse resident peritoneal macrophage. The present studies show that this oxidized LDL does not inhibit the motility of mouse monocytes and actually exhibits a chemotactic activity for human monocytes; the chemotactic activity of the oxidized LDL resides in the lipid fraction. These findings allow us to propose a pathogenetic sequence by which elevated plasma LDL levels, followed by oxidative modification in the arterial wall, could sufficiently account for the generation of the lipid-laden foam cells and the initiation of the fatty streak, the earliest well-defined lesion in atherogenesis.Accumulation of lipid in the arterial intima is central to the development of atherosclerosis. Intimal lipid accumulates intracytoplasmically in foam cells, which are derived both from medial smooth muscle cells (1, 2) and monocyte-derived macrophages (3)(4)(5), the latter probably being quantitatively more important (3-5). Exactly how monocytes are recruited and retained in the artery wall remains unclear, but probably the initial event is adhesion to the endothelial surface (3) followed by penetration that is influenced by a chemotactic factor(s). Many different factors chemotactic for monocytes have been described (6); the relative importance of these remains uncertain. Crude extracts of whole aorta contain chemotactic activity (7), and cultured arterial smooth muscle cells and macrophages release chemotactic activity into the culture medium (8). We recently described release of chemotactic activity for mouse resident peritoneal macrophages from cultured aortic endothelial cells (9) and showed that oxidatively modified low density lipoprotein (LDL) inhibited the chemotactic response of the macrophage. In the present studies we confirm the finding of Berliner et al. (10) that endothelial cell-conditioned medium is chemotactic also for human monocytes. However, oxidatively modified LDL, instead of inhibiting the motility of monocytes actually enhances their motility. We further show that the chemotactic activity resides in the lipid fraction of the modified LDL, presumably in one or another peroxidized lipid component. Thus oxidative modification of LDL, in addition to favoring the accumulation of cholesterol stores in developing foam cells (11), could play a role in recruitment and retention of monocyte/macrophages into the subendothelial space and, finally, may contribute to atherogenesis through injury to endothelial cells. MATERIALS AND METHODSHam's F-10 medium and fetal bovine serum were from HyClone (Logan, UT); female Swiss Webster mice were from Simonsen Laboratories (Gilroy, CA); Ficoll/Hypaque, bovine serum albumin, zymosan A, fucoidi...
Native low density lipoprotein (LDL) does not affect monocyte/macrophage motility. On the other hand, oxidatively modified LDL inhibits the motility of resident peritoneal macrophages yet acts as a chemotactic factor for circulating human monocytes. We now show that lysophosphatidylcholine (lyso-PtdCho), which is generated by a phospholipase A2 activity during LDL oxidation, is a potent chemotactic factor for monocytes. It is not chemotactic for neutrophils or for resident macrophages. Platelet-activating factor, after treatment with phospholipase A2, becomes chemotactic for monocytes, whereas the intact factor is not. Synthetic 1-palmitoyl-lyso-PtdCho showed chemotactic activity comparable to that of the lyso-PtdCho fraction derived from oxidized LDL. The results suggest that Iyso-PtdCho in oxidized LDL may favor recruitment of monocytes into the arterial wall during the early stages of atherogenesis. Generation of lyso-PtdCho, either from LDL itself or from membrane phospholipids of damaged cells, could play a more general role in inflammatory processes throughout the body.One of the characteristics of the earliest atherosclerotic lesion is the intimal accumulation of lipid-laden foam cells derived predominantly from monocyte/macrophages (1-5).Exactly how these cells are recruited and retained in the artery wall remains unclear, but it seems likely that adhesion molecules and chemotactic factors are involved. Although many different chemotactic factors for monocytes have been described (6-9), their relative importance in relation to atherogenesis remains to be determined. The possibility that a high plasma level of low density lipoprotein (LDL) might play a role in monocyte recruitment seemed attractive in view of the fact that a high LDL level is the proximate consequence of LDL receptor deficiency in homozygous familial hypercholesterolemia and, therefore, must somehow account for the genesis of the fatty streak lesions (10
The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein‐coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
Abstract-The purpose of this study was to determine whether superoxide anion is produced endogenously in the rat aortic adventitia and whether sufficient superoxide anion is produced to interfere with the response of the rat aorta to nitric oxide. Relaxation was measured in rings of the rat thoracic aorta, which were oriented so that the adventitial or luminal surface could be preferentially exposed to nitric oxide or sodium nitroprusside. To accomplish this, the rings were mounted (1) with the adventitia facing outward, (2) with the adventitia facing inward after inverting, or (3) with the adventitia facing outward after inverting twice (to control for the inverting procedure). The relaxation to nitric oxide, but not to sodium nitroprusside, was less in rings with the adventitia facing outward compared with those in which it faced inward. In contrast, the response to nitric oxide via either surface was similar when extracellular superoxide anion was scavenged with superoxide dismutase. Incubation of rings with nitro blue tetrazolium (NBT) resulted in blue formazan staining of the adventitia, and lucigenin chemiluminescence was significantly greater when detected from the adventitial compared with the intimal aspect of the artery. The reduction of NBT in intact aortic rings was 30Ϯ2 pmol ⅐ min Ϫ1 ⅐ mg Ϫ1 and was significantly decreased by superoxide dismutase to 19Ϯ2 pmol ⅐ min Ϫ1 ⅐ mg Ϫ1 and by a synthetic superoxide dismutase mimic, Euk-8, to 11Ϯ2 pmol ⅐ min Ϫ1 ⅐ mg Ϫ1. The NADPH oxidase inhibitor, diphenyleneiodonium, decreased NBT reduction to 9Ϯ1 pmol ⅐ min Ϫ1 ⅐ mg Ϫ1, whereas inhibitors of xanthine oxidase, mitochondrial oxidases, and nitric oxide synthase were ineffective. Immunohistochemical staining indicated the localization of NADPH oxidase proteins gp91 phox , p22 phox , p47 phox , and p67 phox almost exclusively in the adventitia of the rat aorta with no substantial staining in the media. These results indicate that NADPH oxidase located in the adventitia of rat thoracic aorta generates sufficient extracellular superoxide anion to constitute a barrier capable of inactivating nitric oxide. This study suggests that adventitial superoxide anion can play a role in the pathophysiology of the arterial wall. (Circ Res. 1998;82:810-818.)
Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22phox and gp91phox, as well as the Ca2+ concentration-responsive gp91phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22phox was not detectable, both gp91phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22phox or gp91phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death.
nant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-B (NF-B). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-B activation and suppress cell proliferation (11), the nature of the enzyme responsible for ROS production in melanoma cells has not been determined. To address this issue, we now have characterized the source of ROS production in melanoma cells. We report that ROS are generated by isolated, cytosol-free melanoma plasma membranes, with inhibition by NAD(P)H oxidase inhibitors. The p22 phox , gp91 phox , and p67 phox components of the human phagocyte NAD(P)H oxidase and the gp91 phox homolog NOX4 were demonstrated in melanomas by RT-PCR and sequencing, and protein product for both p22 phox and gp91 phox was detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22 phox and NOX4. Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22 phox and NOX4. Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive DNA binding of nuclear protein to the NF-B and cAMP-response element consensus oligonucleotides, without affecting DNA binding activity to activator protein-1 or OCT-1. This suggests that ROS generated in autocrine fashion by an NAD(P)H oxidase may play a role in signaling malignant melanoma growth. superoxide anion; diphenylene iodonium; p22 phox ; gp91 phox ; p67 phox ; NOX1; NOX4; nuclear factor-B; cAMP response element; dicumarol REACTIVE OXYGEN SPECIES (ROS) generated by an NAD(P)H oxidase have been recently recognized as important signaling molecules for proliferation of normal cells. The role of a signaling NAD(P)H oxidase has been most extensively explored in vascular smooth muscle cells, where both p22 phox and the unique gp91 phox homolog NOX1 have been shown to be important for function of an NAD(P)H oxidase activity that mediates angiotensin IIinduced superoxide (O 2 Ϫ ) formation and redox-sensitive signaling pathways (19,30). Similar but structurally distinct NAD(P)H oxidases also perform signaling functions in normal vascular endothelial cells (18, 25) and adventitial cells (38), and the gp91 phox homolog NOX4 has recently been described in renal tubular epithelium (17, 45), fetal tissue (12), placenta (12), and proliferating vascular smooth muscle (30).Like normal cells, human tumor cells also produce substantial amounts of ROS spontaneously (15,37,49), and evidence points to a role for these ROS in signaling neoplastic proliferation. Mitogenic signaling through both Ras (22) and Rac (26) is mediated by O 2 Ϫ , and transfection with mitogenic oxidase NOX1 transforms normal fibroblasts (48) and creates cell lines that are tumorigenic in athymic mice (2). The NOX1 homolog has been found expressed in the Caco human colon carcinoma cells (12,28,48) and HepG2 hepatoma cells (28), and gp91 phox expression has been...
Activation of the superoxide generating system in human neutrophils is thought to involve the interaction or assembly of cytochrome b with other cytosolic and membrane proteins. We have now co-isolated by conventional purification procedures a protein of relative molecular mass 22,000 with cytochrome b. This Ras-related protein is not a fragment of either of the subunits of cytochrome b, and its primary structure, as determined by the sequencing of its complementary DNA, is identical to that predicted from a recently cloned ras-related gene, rap1 (also termed Krev-1). Immunoaffinity purification on anti-cytochrome and anti-Ras immunoaffinity matrices indicates an association between cytochrome b and the Ras-related protein. The association of a Ras-related GTP-binding protein with cytochrome b of human neutrophils could indicate a role for such a protein in the transduction, regulation or structure of the superoxide generating system.
Cultured rabbit and bovine aortic endothelial cells generated chemotactic activity for mouse resident peritoneal macrophages, demonstrable in the conditioned medium. This chemotactic activity was heat stable and was not extracted into chloroform/methanol. It was inhibited by addition of endothelial cell-modified low density lipoprotein (EC-modifiled LDL), a form of LDL shown previously to contain peroxidized lipids, increased lysophosphatidylcholine, and partially degraded apoprotein B. The chemotactic activity was also inhibited by LDL previously oxidized in the absence of cells with 5 FAM Cu2 . Inhibitory activity was present in the lipid extract of EC-modified LDL but not in that of native LDL, presumably representing peroxidized lipid components. EC-modified LDL also inhibited the chemotactic activity of zymosan-activated serum. Because EC-modified LDL is taken up in part by way of the acetyl-LDL receptor, the effects of acetyl-LDL were tested. Rather than inhibiting chemotaxis, acetyl LDL showed intrinsic positive chemotactic activity as did also fucoidin and polyinosinic acid, both of which also interact with the acetyl-LDL receptor. These studies suggest mechanisms by which macrophages may be recruited into the subendothelial space by endothelial cell-derived chemotactic factors or by natural polyanions structurally related to fucoidin or polyinosinic acid and then become "trapped" there because of the inhibitory effects of peroxidized lipid components in modified forms of LDL.A growing body of evidence indicates that many or most of the lipid-laden foam cells in the atherosclerotic lesion are derived from monocyte/macrophages (1-3). One of the earliest events in experimental atherosclerosis is the adherence of monocytes to the arterial endothelium and penetration into the subendothelial space (3). The mechanisms by which macrophages take up lipoproteins and store cholesterol have been intensively studied over the past several years. These studies show that the macrophage has only a limited of number of receptors for the specific uptake of native low density lipoprotein (LDL) but that it avidly takes up certain chemically modified forms of LDL via an alternative specific, saturable receptor-the acetyl-LDL receptor (4). We have shown previously that LDL incubated overnight with cultured endothelial cells in F-10 medium (endothelial cell-modified LDL; EC-modified LDL) is biologically modified to a form that is also more rapidly degraded by macrophages, in part by the same acetyl-LDL receptor (5-7). Macrophages also express a specific receptor for a modified form of very low density lipoprotein (VLDL) that accumulates in cholesterol-fed animals, the so-called ,-VLDL receptor (8). Finally, it has been shown that the macrophage secretes lipoprotein lipase, which facilitates uptake and storage of lipids derived from very low density, triglyceriderich lipoproteins (9, 10).It remains unclear what factors lead to the recruitment and retention of the monocyte/macrophage in the artery wall. Many chemotactic ...
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