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Prepublished online as a Blood First Edition Paper on February 13, 2003; DOI 10.1182/blood-2002-09-2711. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-09-2711v1 101/12/4847    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Shatrov, V. A. Articles by Brüne, B. Search for Related Content PubMed PubMed Citation Articles by Shatrov, V. A. Articles by Brüne, B. Related Collections Gene Expression Brief Reports Hemostasis, Thrombosis, and Vascular Biology Phagocytes Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 June 2003, Vol. 101, No. 12, pp. 4847-4849 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Brief report Oxidized low-density lipoprotein (oxLDL) triggers hypoxia-inducible factor-1 (HIF-1) accumulation via redox-dependent mechanisms Vladimir A. Shatrov, Vadim V. Sumbayev, Jie Zhou, and Bernhard Brüne From the Faculty of Biology, Department of Cell Biology, University of Kaiserslautern, Germany.    Abstract Top Abstract Introduction Study design Results and discussion References   Oxidized low-density lipoprotein (oxLDL) and macrophages play a central role in atherosclerosis. Here, we obtained evidence that oxLDL induced hypoxia-inducible factor-1 (HIF-1) protein accumulation in human macrophages (Mono-Mac-6) under normoxia. HIF-1 accumulation was attenuated by pretreatment with the antioxidant N-acetyl-L-cysteine (NAC), the nitric oxide (NO) donor S-nitrosoglutathione (GSNO), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors such as diphenyleniodonium (DPI) or 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), thus implicating the contribution of oxLDL-generated reactive oxygen species (ROS). Whereas oxLDL did not modulate HIF-1 mRNA levels, experiments with cycloheximide pointed to a translational mechanism in oxLDL action. HIF-1–dependent luciferase reporter gene analysis underscored HIF-1 transactivation. Our results indicate that oxLDL induced HIF-1 accumulation and HIF-1–dependent reporter gene activation in human macrophages via a redox-mediated pathway. This finding may suggest a role of HIF-1 in atherosclerosis and oxLDL-induced pathogenesis.    Introduction Top Abstract Introduction Study design Results and discussion References   Oxidatively modified low-density lipoprotein (oxLDL) emerged as a pathogenetic factor in atherosclerosis.1 There is accumulating evidence for oxidative stress in vascular dysfunction/atherogenesis and a significant contribution of macrophages in oxLDL uptake, foam cell formation, and disease progression.1 Considering the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in generating (reactive oxygen species) ROS, we recently demonstrated that NADPH oxidase is a major source of ROS in response to oxLDL with the further notion that diphenyleniodonium (DPI), a NADPH oxidase inhibitor, eliminated ROS generation in macrophages.2 Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor composed of and subunits.3 Although HIF-1 is constitutively expressed in many cell types, HIF-1 is present at undetectable amounts under normoxia because of rapid proteasomeal degradation and is stabilized on hypoxia.4,5 Following HIF-1/HIF-1 heterodimerization and translocation to the nucleus, binding to promoter-specific HRE (hypoxia response element) sites drives classical hypoxia responsive genes.6,7 Studies suggested that ROS provides a redox signal for hypoxic HIF-1 induction.8 Moreover, ROS appears to regulate HIF-1 under normoxia. In some cell types, increased ROS production has been associated with HIF-1 activation by hormones, growth factors, and transition metals.8,9 In addition, direct exposure of cells to ROS may stabilize HIF-1 and promote vascular endothelial growth factor (VEGF) expression.8,10 Along that line cytokine-mediated regulation of HIF-1 may require a nonhypoxic but ROS-sensitive pathway.11 Here, we showed that oxLDL provoked HIF-1 accumulation and HIF-1–dependent transcriptional activation in human Mono-Mac-6 (MM6) macrophages through a ROS-dependent pathway.    Study design Top Abstract Introduction Study design Results and discussion References   Cell cultures MM6 cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, 9 µg/mL bovine insulin and antibiotics in a humidified 5% CO2 atmosphere at 37°C. Cell dishes at 80% to 90% confluence were exposed to native low-density lipoprotein (nLDL), oxLDL, and/or inhibitors. RCC4 cells, which constitutively express HIF-1 were kindly provided by Prof Ratcliffe, Wellcome Trust Centre for Human Genetics, University of Oxford, United Kingdom.12,13 nLDL was purchased from Sigma (Taufkirchen, Germany) and oxidized as described.2 Western blot analysis HIF-1 (antibody from Becton Dickinson, Heidelberg, Germany) as well as actin (to confirm equal protein loading) was determined by Western analysis as described.14 Transient transfection MM6 cells (5 x 105) were seeded in 6-cm dishes 1 day before transfection with 3 µg plasmid pGL3-EPO-HRE-SV40-LUC, which harbors 3 copies of the erythropoietin hypoxia responsive element in front of the SV 40 promoter or a VEGF reporter plasmid (pGL3-1.5kbVEGFprom, obtained from Prof Maity, University of Pennsylvania Medical Center, Philadelphia), for 8 hours, using dioleoyl-1,2-diacyl-3-trimethylammonium-propane (DOTAP) for transfection according to company instructions. Thereafter, medium was changed and cells were stimulated. RT-PCR MM6 cells (5 x 106) were incubated with or without 200 µg/mL oxLDL for 5 hours. Total RNA was isolated using the peqGOLD RNAPure kit, followed by HIF-1 mRNA reverse transcriptase–polymerase chain reaction (RT-PCR). Primer selection was as follows: HIF-1,5'-CTCAAAGTCGGACAGCCTCA-3',5'-CCCTGCAGTAGGTTTCTGCT-3'; actin, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3', 5'-CTAGAAGCATTTGCGGTCGACGATGGAGGG-3'. Amplification program was as follows: 95°C, 30 seconds; 56°C, 30 seconds; 72°C, 1 minute; 20 cycles; 72°C, 10 minutes. Products were separated on 2% agarose gels and visualized with ethidium bromide. Statistical analysis Each experiment was performed at least 3 times, and representative data are shown. Data are presented as means ± SD. The unpaired Student t test was used to evaluate the significance of differences between groups, accepting P < .05 as the level of significance.    Results and discussion Top Abstract Introduction Study design Results and discussion References   To examine the effect of oxLDL on HIF-1 accumulation, MM6 cells were treated with oxLDL or nLDL. As shown in Figure 1A, oxLDL dose-dependently (25-200 µg/mL) induced HIF-1 accumulation. oxLDL was obtained by the CuSO4/EDTA oxidation protocol. Therefore, we ruled out that neither CuSO4 nor EDTA at the relevant concentrations promoted HIF-1 accumulation (Figure 1A). As a positive control, to unequivocally localize HIF-1, we used an RCC4 protein lysate with constitutive HIF-1 expression (Figure 1A). Furthermore, we established that oxLDL but not nLDL, induced HIF-1 accumulation (Figure 1B). The most efficient response was noticed between 4 and 8 hours. Under these experimental conditions oxLDL was nontoxic (data not shown). View larger version (36K): [in this window] [in a new window]   Figure 1.. HIF-1 accumulation in response to oxLDL. (A) MM6 cells were treated with increasing concentrations of oxLDL for 8 hours. For control reasons, 2 µM CuSO4, 15 µM EDTA (ethylenediaminetetraacetic acid), and a lysate of RCC4 cells were processed in parallel. (B) MM6 cells were time-dependently stimulated with 200 µg/mL oxLDL or nLDL. (C) MM6 cells were pretreated with 3 mM NAC, 30 µM DPI, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) for 2 hours, followed by stimulation with 200 µg/mL oxLDL for 8 hours. S-nitrosoglutathione (GSNO; 1 mM) was supplied together with oxLDL for 8 hours. (D) Cells were pretreated with 30 µM PD98059 or 30 µM SB203580 for 2 hours, following by stimulation with 200 µM oxLDL for 8 hours. (E) HIF-1 expression was induced by exposing MM6 cells to 200 µg/mL oxLDL for 8 hours. Cycloheximide (CHX) at a final concentration of 10 µg/mL was added for the times indicated (15-60 minutes). (F) Cells were treated with 200 µg/mL oxLDL for 5 hours, and HIF-1 mRNA was followed by RT-PCR analysis as described. All experiments were performed at least 3 times and representative data are shown. HIF-1 protein was detected by Western blot analysis as outlined in "Study design."   Reports from several groups suggested that ROS stabilizes HIF-1 and activates HIF-1.8-10 Therefore, we investigated whether ROS, produced in response to oxLDL, contributed to HIF-1 accumulation (Figure 1C). N-acetyl-L-cysteine (NAC) is an antioxidant, known to decrease ROS. The addition of 3 mM NAC to MM6 cells for 2 hours prior to the stimulation with 200 µg/mL oxLDL for 8 hours completely blocked HIF-1 accumulation. DPI inhibits oxLDL-mediated ROS formation in macrophages by blocking NADPH oxidase.2 When cells were pretreated with 30 µM DPI for 2 hours prior to oxLDL stimulation, HIF-1 accumulation was suppressed. Because DPI may inhibit mitochondrial complex I, we studied the effect of the structurally unrelated NADPH oxidase inhibitor AEBSF in parallel, which again prevented HIF-1 accumulation. The effect of nitric oxide (NO) derived from the most physiologic donor GSNO may be rationalized by the diffusion controlled interaction of with NO, thus allowing NO to attenuate HIF-1 accumulation. Our experiments with NADPH oxidase inhibitors (DPI, AEBSF) and GSNO suggest that might play a role in oxLDL-induced HIF-1 accumulation. Previously, it was demonstrated that mitogen-activated protein kinase (MAPK) family members such as p38 or ERK1/2 attenuated transactivation of HIF-1 in hypoxic cells. In MM6 cells, neither p38 (SB203580) nor ERK (PD98059) inhibitors affect oxLDL-induced HIF-1 accumulation (Figure 1D). Both inhibitors did not significantly alter HIF-1 transactivation in response to oxLDL (data not shown). Next, we examined the time-dependent disappearance of HIF-1 in the presence of the translational inhibitor cycloheximide (Figure 1E). Cells were treated with oxLDL for 4 hours followed by the subsequent addition of cycloheximide versus vehicle for 15 to 60 minutes. Cycloheximide decreased HIF-1 expression starting at 15 minutes, suggesting that oxLDL increased HIF-1 through a translational mechanism. The persistence of detectable HIF-1 protein 15 minutes following cycloheximide treatment could represent a delay in the onset of the cycloheximide effect but implies that the mode of action of oxLDL leaves the degradative pathway largely intact. Thus, it appeared advised to examine HIF-1 mRNA expression. RT-PCR (Figure 1F) revealed that oxLDL left HIF-1 mRNA levels unaltered. We then tested the ability of oxLDL to activate a HIF-1–dependent luciferase reporter gene construct, containing 3 copies of a hypoxia-response element derived from the human erythropoietin gene. Treatment of MM6 cells with oxLDL resulted in significant reporter gene activation that was largely abrogated by DPI (Figure 2A). nLDL showed no reporter signal, whereas hypoxia elicited a luciferase activity comparable to oxLDL. Similarly, treatment of cells with oxLDL led to an increase in VEGF promoter activity (Figure 2B). Our results are consistent with previous studies, showing a 2- to 3-fold increase in reporter activity by hypoxia14,15 and further studies reporting hypoxic VEGF production in macrophages.16 Atherosclerosis is a progressive disease and a common cause of morbidity and mortality in the Western world. Therefore, our results on HIF-1 accumulation by oxLDL appear of clinical relevance. It is well established that HIF-1 plays a major role in VEGF expression and angiogenesis9 with the notion that VEGF mediates important alterations associated with atherogenesis and the angiogenic activity of macrophages.16 Recent findings suggest that neovascularization within atherosclerotic plaques is a sign of advanced atherosclerosis/restenosis.17 It is tempting to speculate that oxLDL-induced HIF-1 accumulation and VEGF up-regulation are important in these pathophysiologic settings. A series of molecules and molecular pathways emerged that contribute to the pathogenesis and progression of both atherosclerosis and cancer.18,19 HIF-1 may be added to that list, considering the important tumor angiogenic role of HIF-1. Our observations propose a new signaling quality of oxLDL in HIF-1 accumulation and demonstrate ROS involvement. This appears of considerable importance to fully understand progression of atherosclerosis and remodeling of the arterial wall by oxLDL. View larger version (22K): [in this window] [in a new window]   Figure 2.. HIF-1–dependent reporter gene activation by oxLDL. MM6 cells were transiently transfected with a luciferase reporter gene construct, containing (A) 3 copies of the HRE of the erythropoietin gene (pGL3-EPO-HRE-SV40-LUC) or (B) pGL3-1.5kbVEGFprom. Transfected cells were stimulated for 8 hours with 200 µg/mL oxLDL or nLDL, in the absence or presence of 30 µM DPI, or exposed for 8 hours to hypoxia (1% oxygen) followed by luciferase activity determination. Luciferase activity is expressed relative to controls set as 100%. Values represent the fold induction of 4 independent experiments.      Footnotes   Submitted September 5, 2002; accepted February 3, 2003. Prepublished online as Blood First Edition Paper, February 13, 2003; DOI 10.1182/blood-2002-09-2711. Supported by funds of the Deutsche Forschungsgemeinschaft (DFG) (BR 999), European Commission (EC) (QLK 6-GT-2000-64), and VerUm Foundation. V.A.S. is a research fellow of the Alexander von Humbolt Foundation. V.A.S. and V.V.S. contributed equally to this study. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Bernhard Brüne, Department of Cell Biology, University of Kaiserslautern, Erwin-Schroedinger-Strasse, 67663 Kaiserslautern, Germany; e-mail: bruene{at}rhrk.uni-kl.de.    References Top Abstract Introduction Study design Results and discussion References   Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem. 1997;272: 20963-20966.[Free Full Text] Fischer B, von Knethen A, Brüne B. Dualism of oxidized lipoproteins in provoking and attenuating the oxidative burst in macrophages: role of peroxisome proliferator-activated receptor-gamma. J Immunol. 2002;168: 2828-2834.[Abstract/Free Full Text] Wang GL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem. 1995;270: 1230-1237.[Abstract/Free Full Text] Salceda S, Caro J. Hypoxia-inducible factor 1 (HIF-1) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem. 1997;272: 22642-22647.[Abstract/Free Full Text] Huang LE, Gu J, Schau M, Bunn HF. Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A. 1998;95: 7987-7992.[Abstract/Free Full Text] Hellwig-Burgel T, Rutkowski K, Metzen E, Fandrey J, Jelkmann W. Interleukin-1 and tumor necrosis factor- stimulate DNA binding of hypoxia-inducible factor-1. Blood. 1999;94: 1561-1567.[Abstract/Free Full Text] Faquin WC, Schneider TJ, Goldberg MA. Effect of inflammatory cytokines on hypoxia-induced erythropoietin production. Blood. 1992;79: 1987-1994.[Abstract/Free Full Text] Chandel NS, McClintock DS, Feliciano CE, et al. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1 during hypoxia. A mechanism of O2 sensing. J Biol Chem. 2000;275: 25130-25138.[Abstract/Free Full Text] Richard DE, Berra E, Pouyssegur J. Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1 in vascular smooth muscle cells. J Biol Chem. 2001;275: 26765-26771.[Abstract/Free Full Text] Brauchle M, Funk JO, Kind P, Werner S. Ultraviolet B and H2O2 are potent inducers of vascular endothelial growth factor expression in cultured keratinocytes. J Biol Chem. 1996;271: 21793-21797.[Abstract/Free Full Text] Haddad JJ, Land SC. A non-hypoxic, ROS-sensitive pathway mediates TNF-alpha-dependent regulation of HIF-1alpha. FEBS Lett. 2001;505: 269-274.[CrossRef][Medline] [Order article via Infotrieve] Maxwell PH, Wiesener MS, Chang G-W, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999;399: 271-275.[CrossRef][Medline] [Order article via Infotrieve] Wykoff CC, Pugh CW, Maxwell PH, Harris AL, Ratcliffe PJ. Identification of novel hypoxia dependent and independent target genes of the von Hippel-Lindau (VHL) tumour suppressor by mRNA differential expression profiling. Oncogene. 2000;19: 6297-6305.[CrossRef][Medline] [Order article via Infotrieve] Sandau KB, Zhou J, Kietzmann T, Brüne B. Regulation of the hypoxia-inducible factor 1alpha by the inflammatory mediators nitric oxide and tumor necrosis factor-alpha in contrast to desferroxamine and phenylarsine oxide. J Biol Chem. 2001;276: 39805-39811.[Abstract/Free Full Text] Maity A, Pore N, Lee J, Solomon D, O'Rourke DM. Epidermal growth factor receptor transcriptionally up-regulates vascular endothelial growth factor expression in human glioblastoma cells via a pathway involving phosphatidylinositol 3' kinase and distinct from that induced by hypoxia. Cancer Res. 2000;60: 5879-5886.[Abstract/Free Full Text] Xiong M, Elson G, Legarda D, Leibovich SJ. Production of vascular endothelial growth factor by murine macrophages: regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway. Am J Pathol. 1998;153: 587-598.[Abstract/Free Full Text] Laukkanen J, Ylä-Herttuala S. Genes involved in atherosclerosis. Exp Nephrol. 2002;10: 150-163.[CrossRef][Medline] [Order article via Infotrieve] Ross JS, Stagliano NE, Donovan MJ, Breitbart RE, Ginsburg GS. Atherosclerosis and cancer: common molecular pathways of disease development and progression. Ann N Y Acad Sci. 2001;947: 271-293.[Abstract/Free Full Text] Toi M. Bando H, Ogawa T, Muta M, Hornig C, Weich HA. Significance of vascular endothelial growth factor (VEGF)/soluble VEGF receptor-1 relationship in breast cancer. Int J Cancer. 2002;98: 14-18.[CrossRef][Medline] [Order article via Infotrieve] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: M. Mayr, A. Sidibe, and A. Zampetaki The Paradox of Hypoxic and Oxidative Stress in Atherosclerosis J. Am. Coll. Cardiol., April 1, 2008; 51(13): 1266 - 1267. [Full Text] [PDF] T. Oda, K. Hirota, K. Nishi, S. Takabuchi, S. Oda, H. Yamada, T. Arai, K. Fukuda, T. Kita, T. Adachi, et al. Activation of hypoxia-inducible factor 1 during macrophage differentiation Am J Physiol Cell Physiol, July 1, 2006; 291(1): C104 - C113. [Abstract] [Full Text] [PDF] H. J. Knowles, D. R. Mole, P. J. Ratcliffe, and A. L. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-09-2711v1 101/12/4847    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Shatrov, V. A. Articles by Brüne, B. Search for Related Content PubMed PubMed Citation Articles by Shatrov, V. A. Articles by Brüne, B. Related Collections Gene Expression Brief Reports Hemostasis, Thrombosis, and Vascular Biology Phagocytes Social Bookmarking                   What's this?

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