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• W2003753810 abstract "Activation of the lectin-like oxLDL receptor (LOX-1) promotes atherosclerosis. Oxidized LDL (oxLDL) increases production of reactive oxygen species (ROS) and leads to the development of endothelial dysfunction. The molecular causes for oxLDL to induce oxidative DNA damage and metabolic dysfunction remain uncertain. Here we report treatment of cultured human coronary arterial endothelial cells (HCAEC) with oxLDL to cause oxidative DNA damage as determined by a 3-fold increase in 8-OH-desoxyguanosine adduct formation and a 4-fold induction of the growth arrest and DNA damage-inducible transcripts GADD45 and GADD153. Oxidative stress resulted in activation of Oct-1, a transcriptional repressor of various vascular cytochrome P450 (CYP) monooxygenases. Activation of Oct-1 was protein kinase C (PKC)-mediated. Binding of Oct-1 to promoter sequences of CYP monooxygenases was increased upon treatment of HCAEC with oxLDL. This resulted in repressed production of endothelium-derived hyperpolarization factor 11,12-epoxyeicosatrieonic acid. Small interference RNA-mediated functional knockdown of Oct-1 prevented oxLDL-mediated silencing of CYP expression. Inhibition of LOX-1 attenuated oxLDL-mediated endothelial DNA damage, Oct-1/DNA binding, and reversed impaired production of EDHF. Taken collectively, oxLDL induced oxidative DNA damage and activation of Oct-1 to result in metabolic dysfunction of coronary arterial endothelium. Activation of the lectin-like oxLDL receptor (LOX-1) promotes atherosclerosis. Oxidized LDL (oxLDL) increases production of reactive oxygen species (ROS) and leads to the development of endothelial dysfunction. The molecular causes for oxLDL to induce oxidative DNA damage and metabolic dysfunction remain uncertain. Here we report treatment of cultured human coronary arterial endothelial cells (HCAEC) with oxLDL to cause oxidative DNA damage as determined by a 3-fold increase in 8-OH-desoxyguanosine adduct formation and a 4-fold induction of the growth arrest and DNA damage-inducible transcripts GADD45 and GADD153. Oxidative stress resulted in activation of Oct-1, a transcriptional repressor of various vascular cytochrome P450 (CYP) monooxygenases. Activation of Oct-1 was protein kinase C (PKC)-mediated. Binding of Oct-1 to promoter sequences of CYP monooxygenases was increased upon treatment of HCAEC with oxLDL. This resulted in repressed production of endothelium-derived hyperpolarization factor 11,12-epoxyeicosatrieonic acid. Small interference RNA-mediated functional knockdown of Oct-1 prevented oxLDL-mediated silencing of CYP expression. Inhibition of LOX-1 attenuated oxLDL-mediated endothelial DNA damage, Oct-1/DNA binding, and reversed impaired production of EDHF. Taken collectively, oxLDL induced oxidative DNA damage and activation of Oct-1 to result in metabolic dysfunction of coronary arterial endothelium. Oxidized low density lipoproteins (oxLDL) 2The abbreviations used are: oxLDL, oxidized low density lipoprotein; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; siRNA, small interference RNA; apoE, apolipoprotein E; 8-OH-dG, 8-hydroxy-2′-deoxyguanosine; 11,12-EET, 11,12-epoxyeicosatrienoic acid; EDHF, endothelial-derived hyperpolarization factor; HCAEC, human coronary arterial endothelial cells; ROS, reactive oxygen species; PKC, protein kinase C; moxLDL, mildly oxidized LDL; dG, deoxyguanosine; vWF, von Willebrandt factor. 2The abbreviations used are: oxLDL, oxidized low density lipoprotein; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; siRNA, small interference RNA; apoE, apolipoprotein E; 8-OH-dG, 8-hydroxy-2′-deoxyguanosine; 11,12-EET, 11,12-epoxyeicosatrienoic acid; EDHF, endothelial-derived hyperpolarization factor; HCAEC, human coronary arterial endothelial cells; ROS, reactive oxygen species; PKC, protein kinase C; moxLDL, mildly oxidized LDL; dG, deoxyguanosine; vWF, von Willebrandt factor. are atherogenic and considered to be a fundamental risk factor in the initiation and progression of atherosclerosis (1Suzuki T. Kohno H. Hasegawa A. Toshima S. Amaki T. Kurabayashi M. Nagai R. Suzuki T. Amaki T. Nagai R. Hasegawa A. Toshima S. Kurabayashi M.H. Shimada K. Nakamura H. Teramoto T. Yamaguchi H. Nishiyama S. Takahashi H. Michishita I. Sugano Z. Konoshi K. Clin. Biochem. 2002; 35: 347-353Crossref PubMed Scopus (57) Google Scholar). Specifically, uptake of oxLDL into the endothelium is mediated through interaction with the lectin-like oxLDL receptor (LOX-1). This receptor was reported to be overexpressed in human atherosclerotic lesions (2Kataoka H. Kume N. Miyamoto S. Minami M. Moriwaki H. Murase T. Sawamura T. Masaki T. Hashimoto N. Kita T. Circulation. 1999; 99: 3110-3117Crossref PubMed Scopus (396) Google Scholar). Transgenic overexpression of LOX-1 in an apoE-deficient background increases atherosclerotic plaque incidence (3Inoue K. Arai Y. Kurihara H. Kita T. Sawamura T. Circ. Res. 2005; 97: 176-184Crossref PubMed Scopus (155) Google Scholar), and LOX-1 expression is highly correlated with plaque instability (4Ishino S. Mukai T. Kume N. Asano D. Ogawa M. Kuge Y. Minami M. Kita T. Shiomi M. Saji H. Atherosclerosis. 2007; 195: 48-56Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In strong contrast, deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed a high cholesterol diet (5Mehta J.L. Sanada N. Hu C.P. Chen J. Dandapat A. Sugawara F. Satoh H. Inoue K. Kawase Y. Jishage K. Suzuki H. Takeya M. Schnackenberg L. Beger R. Hermonat P.L. Thomas M. Sawamura T. Circ. Res. 2007; 100: 1634-1642Crossref PubMed Scopus (372) Google Scholar). Furthermore, human genetic studies link LOX-1 polymorphisms to cardiovascular disease susceptibility (6Mango R. Biocca S. del Vecchio F. Clementi F. Sangiuolo F. Amati F. Filareto A. Grelli S. Spitalieri P. Filesi I. Favalli C. Lauro R. Mehta J.L. Romeo F. Novelli G. Circ. Res. 2005; 97: 152-158Crossref PubMed Scopus (103) Google Scholar).Mechanistically, oxLDL fosters intracellular production of reactive oxygen species (ROS) (7Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Aiba Y. Tanaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1155) Google Scholar, 8Cominacini L. Garbin U. Pasini A.F. Davoli A. Campagnola M. Pastorino A.M. Gaviraghi G. Lo Cascio V. J. Hypertension. 1998; 16: 1913-1919Crossref PubMed Scopus (86) Google Scholar, 9Thum T. Borlak J. Circ. Res. 2004; 94: 1-13Crossref PubMed Google Scholar) and promotes apoptosis (10Chen J. Mehta J.L. Haider N. Zhang X. Narula J. Li D. Circ. Res. 2004; 94: 370-376Crossref PubMed Scopus (239) Google Scholar). There is also evidence for ROS to cause oxidative DNA damage in endothelium (11Wei Y.H. Lu C.Y. Lee H.C. Pang C.Y. Ma Y.S. Ann. N. Y. Acad. Sci. 1998; 854: 155-170Crossref PubMed Scopus (224) Google Scholar, 12Beckman K.B. Ames B.N. J. Biol. Chem. 1997; 272: 19633-19636Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar), which contributes to the pathogenesis of atherosclerosis and to instability of plaques (13Cai H. Harrison D.G. Circ. Res. 2000; 87: 840-844Crossref PubMed Scopus (3138) Google Scholar, 14Bennett M.R. Circ. Res. 2001; 88: 648-650Crossref PubMed Scopus (82) Google Scholar, 15Lee S.H. Blair I.A. Trends Cardiovasc Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar). In patients with cardiovascular disease, the levels of oxLDL and circulating 8-hydroxy-2′-deoxyguanosine (8-OH-dG) DNA-adducts correlate well (16Inoue T. Inoue K. Maeda H. Takayanagi K. Morooka S. Clin. Chim. Acta. 2001; 305: 115-121Crossref PubMed Scopus (36) Google Scholar). Indeed, 8-OH-dG is an established marker for oxidative DNA damage (17Taddei F. Hayakawa H. Bouton M. Cirinesi A. Matic I. Sekiguchi M. Radman M. Science. 1997; 278: 128-130Crossref PubMed Scopus (245) Google Scholar), and 8-OH-dG is elevated in human atherosclerotic plaques of carotid endarterectomy specimens (18Martinet W. Knaapen M.W. De Meyer G.R. Herman A.G. Kockx M.M. Circulation. 2002; 106: 927-932Crossref PubMed Scopus (364) Google Scholar).Specifically, the cellular responses to DNA damage may include regulation of transcription factors, notably, c-Jun (19Jang J.H. Surh Y.J. Ann. N. Y. Acad. Sci. 2002; 973: 228-236Crossref PubMed Scopus (53) Google Scholar), p53 (20Nicol C.J. Harrison M.L. Laposa R.R. Gimelshtein I.L. Wells P.G. Nat. Genet. 1995; 10: 181-187Crossref PubMed Scopus (214) Google Scholar), and NFκB (21Klaunig J.E. Kamendulis L.M. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 239-267Crossref PubMed Scopus (1259) Google Scholar). More recently, the homeodomain transcription factor Oct-1 was added to the growing list of transcription factors regulated upon DNA damage (22Zhao H. Jin S. Fan F. Fan W. Tong T. Zhan Q. Cancer Res. 2000; 60: 6276-6280PubMed Google Scholar). There is evidence for Oct-1 to play an essential role in vascular biology and was found to be up-regulated in dedifferentiated endothelium (23Thum T. Haverich A. Borlak J. FASEB J. 2000; 14: 740-751Crossref PubMed Scopus (62) Google Scholar). Furthermore, Oct-1 was shown to regulate, at least in part, LOX-1 receptor activity (24Chen J. Liu Y. Liu H. Hermonat P.L. Mehta J.L. Biochem. J. 2006; 393: 255-265Crossref PubMed Scopus (44) Google Scholar, 25Fadel B.M. Boutet S.C. Quertermous T. J. Biol. Chem. 1999; 274: 20376-20383Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 26Schwachtgen J.L. Remacle J.E. Janel N. Brys R. Huylebroeck D. Meyer D. Kerbiriou-Nabias D. Blood. 1998; 92: 1247-1258Crossref PubMed Google Scholar).As of today, the role of Oct-1 in oxLDL-induced vascular injury is uncertain. This transcription factor recognizes an octamer sequence (ATGTAAAT) in promoters of target genes (27Groenen M.A. Dijkhof R.J. van der Poel J.J. van Diggelen R. Verstege E. Nucleic Acids Res. 1992; 20: 4311-4318Crossref PubMed Scopus (51) Google Scholar), which encode proteins for the regulation of development, immune response, cellular repair, and general metabolism (25Fadel B.M. Boutet S.C. Quertermous T. J. Biol. Chem. 1999; 274: 20376-20383Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 26Schwachtgen J.L. Remacle J.E. Janel N. Brys R. Huylebroeck D. Meyer D. Kerbiriou-Nabias D. Blood. 1998; 92: 1247-1258Crossref PubMed Google Scholar, 27Groenen M.A. Dijkhof R.J. van der Poel J.J. van Diggelen R. Verstege E. Nucleic Acids Res. 1992; 20: 4311-4318Crossref PubMed Scopus (51) Google Scholar, 28Rundlof A.K. Carlsten M. Arner E.S. J. Biol. Chem. 2001; 276: 30542-30551Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 29Phillips K. Luisi B. J. Mol. Biol. 2000; 302: 1023-1039Crossref PubMed Scopus (197) Google Scholar, 30Schubart D.B. Rolink A. Kosco-Vilbois M.H. Botteri F. Matthias P. Nature. 1996; 383: 538-542Crossref PubMed Scopus (244) Google Scholar, 31Zheng L. Roeder R.G. Luo Y. Cell. 2003; 114: 255-266Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Previous studies are suggestive for Oct-1 to function as a repressor for certain endothelial-expressed enzymes, such as cytochrome P450 epoxygenases (23Thum T. Haverich A. Borlak J. FASEB J. 2000; 14: 740-751Crossref PubMed Scopus (62) Google Scholar, 32Sterling K. Bresnick E. Mol. Pharmacol. 1996; 49: 329-337PubMed Google Scholar). These epoxygenases catalyze production of endogenous vasoactive molecules, including epoxy fatty acids, of which 11,12-epoxyeicosatrienoic acid (11,12-EET) was reported to be an endothelial-derived hyperpolarization factor (EDHF) (9Thum T. Borlak J. Circ. Res. 2004; 94: 1-13Crossref PubMed Google Scholar, 33Fleming I. Pharmacol. Res. 2004; 49: 525-533Crossref PubMed Scopus (122) Google Scholar). Oct-1 may therefore provide a missing link between oxidative DNA damage and impaired metabolic function of endothelium upon exposure to oxidized lipoproteins.Here, we studied the effects of oxLDL on oxidative DNA damage and Oct-1 expression in cultures of human coronary arterial endothelial cells (HCAEC). We evaluated whether Oct-1 up-regulation after oxidative stress may be mechanistically linked to repressed CYP monooxygenases activity and EDHF production. We also studied whether LOX-1 blockade would prevent DNA damage and reverse metabolic impairment of HCAECs when exposed to oxLDL.EXPERIMENTAL PROCEDURESMaterials—HCAECs and EGM-2MV medium were obtained from Clonetics (San Diego, CA). Human atherosclerotic aortae were obtained from cardiac explants (34Thum T. Borlak J. Lancet. 2000; 355: 979-983Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Healthy control aortic protein extracts were obtained from Biocat (Heidelberg, Germany). In addition, atherosclerotic aortae were explanted from apoE knockout mice fed with a high lipid diet for >4 months, as well as from healthy control mice (C57/BL6, Harlan-Winkelmann, Borchen, Germany). The total RNA Isolation system and the NucleoSpin Tissue kit were from Macherey-Nagel (Düren, Germany). Aprotinin, deoxyguanosine (dG), hydrolyzed samples, 2′-deoxyguanosine (dG), and 8-OH-dG standards were from Sigma. Rotiblock, skim milk powder, and Tween-20 was from Roth. Oct-1 antibody was obtained from Santa Cruz Biotechnology (Heidelberg, Germany) and the anti-rabbit antibody from Chemicon (Hofheim, Germany). [32P]ATP and Microspin G-25 columns were purchased from Amersham Biosciences (München, Germany). T4 polynucleotide kinase was from New England Biolabs (Frankfurt am Main, Germany). Pefabloc was from Roche Applied Science (Ingelheim am Rhein, Germany). Bovine serum albumin was from PAA (Linz, Austria).Preparation of LDL, oxLDL, and moxLDL—LDL was isolated from human plasma by sequential gradient ultracentrifugation as described previously (9Thum T. Borlak J. Circ. Res. 2004; 94: 1-13Crossref PubMed Google Scholar). Oxidized LDL was prepared by incubation of LDL with 5 μmol of CuSO4 for 24 h at 37 °C. moxLDL was prepared by incubation with 10 μmol of Fe2+(SO4) for 24 h at 37 °C as described (34Thum T. Borlak J. Lancet. 2000; 355: 979-983Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Oxidation was monitored using the thiobarbituric acid-reactive substances (TBARS) assay with tetraethoxypropane as an internal standard (9Thum T. Borlak J. Circ. Res. 2004; 94: 1-13Crossref PubMed Google Scholar). Oxidation was stopped by addition of butylated hydroxytoluene (100 μm). Note, the level of oxidation strongly differed between fully (Cu2+)-oxidized LDL (35 nm malonedialdeyde/mg protein) versus minimally (Fe2+)-oxidized LDL (3 nm malonedialdeyde/mg protein). For comparison the TBARS assay revealed no detectable concentration of malonedialdeyde in native LDL preparations.Cell Culture—Primary HCAECs were cultured in 75-cm2 plastic flasks in EGM-2MV medium. Confluent cultures were detached by trypsin/EDTA and plated on 6 wells until 90% confluence was reached. Cultured endothelial cells (fourth passage) were checked by reverse phase contrast microscopy before and after treatment with oxLDL (magnification 20-fold). We additionally examined expression of the endothelial-specific surface protein PECAM-1 to control for cellular differentiation using fluorescence-activated flow cytometry, as described previously (23Thum T. Haverich A. Borlak J. FASEB J. 2000; 14: 740-751Crossref PubMed Scopus (62) Google Scholar).Endothelial cells were treated with increasing doses of oxLDL (10–100 μg/ml) for 24 h. Dose selection was based on published and clinically confirmed oxLDL plasma levels (36Holvoet P. Vanhaecke J. Janssens S. Van de Werf F. Collen D. Circulation. 1998; 98: 1487-1494Crossref PubMed Scopus (594) Google Scholar). As a positive control for the induction of DNA damage, we additionally subjected HCAECs in PBS to UV-C irradiation (wavelength, 254 nm; dose, 10–50 J/m-2), as described (37Tong T. Fan W. Zhao H. Jin S. Fan F. Blanck P. Alomo I. Rajasekaran B. Liu Y. Holbrook N.J. Zhan Q. Exp. Cell Res. 2001; 269: 64-72Crossref PubMed Scopus (56) Google Scholar). Then medium was changed from PBS to EGM-2MV, and 8 h later, Oct-1 expression levels were determined as described below.RNA and cDNA—RNA was isolated from endothelial cells using a total RNA Isolation system according to the manufacturer's recommendation. Quality and quantity of isolated RNA was checked using capillary electrophoresis (Bioanalyzer 2100, Agilent Technologies, Waldbronn, Germany) following the manufacturer's instructions. 2 μg of total RNA from each sample were used for reverse transcription, as described previously (34Thum T. Borlak J. Lancet. 2000; 355: 979-983Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The resulting cDNA was frozen at -20 °C until further experimentation.Real-time and Semi-quantitative PCR—Real-time RT-PCR was done with the Lightcycler (Roche Applied Science, Mannheim, Germany), as described (23Thum T. Haverich A. Borlak J. FASEB J. 2000; 14: 740-751Crossref PubMed Scopus (62) Google Scholar). After an initial denaturation step at 95 °C for 30 s, the PCR reaction was initiated with an annealing temperature of 55 °C for 8 s followed by an extension phase for 8 s (GADD45 and GADD153) or 14 s (Oct-1) at 72 °C and a denaturation cycle at 95 °C for 1 s. The PCR reaction was stopped after a total of 40 cycles, and at the end of each extension phase, fluorescence was observed and used for quantitative measurements within the linear range of amplification. Oligonucleotide sequences were 5′-tcagcgcacgatcactgtc (forward primer) and 5′-ccagcaggcacaacaccac (reverse primer) for detection of the GADD45 gene, 5′-tgaccctgcttctctggctt (forward primer) and 5′-ctgggaggtgcttgtgacct (reverse primer) for the GADD153 gene and 5′-gaatcaacccaccaagcagt (forward primer) and 5′-ggagtggaggtggtctgtgt (reverse primer) for the Oct-1 gene. Exact quantification was achieved by a serial dilution with cDNA produced from endothelial total RNA extracts using 1:5 dilution steps. Gene expression levels were then given as the ratio of the gene of interest (nominator) versus a stable expressed housekeeping gene (cyclophilin A, denominator) (9Thum T. Borlak J. Circ. Res. 2004; 94: 1-13Crossref PubMed Google Scholar, 38Schmid H. Cohen C.D. Henger A. Irrgang S. Schlondorff D. Kretzler M. Kidney Int. 2003; 64: 356-360Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar).Semi-quantitative gene expression of CYP1A1, CYP2B6, CYP2C8, CYP2C9, and CYP2J2 was performed and analyzed as previously described (Ref. 9Thum T. Borlak J. Circ. Res. 2004; 94: 1-13Crossref PubMed Google Scholar and supplemental Table S2).Quantitative Measurement of 7,8-Dihydro-8-oxo-2′-deoxyguanosine—Genomic DNA was extracted from cultured HCAECs treated with oxLDL (10–100 μg/ml) or oxLDL (100 μg/ml) + κ-carrageenan (250 μm), as well as from appropriate controls using the NucleoSpin Tissue kit. DNA (90 μl) was digested with nuclease P1 (5 μg), zinc chloride (3 μl, 20 mmol), and HCl (3 μl, 200 mmol) for 30 min at 45 °C. Then, Tris buffer (3 μl, 500mmol) and 2 μl of alkaline phosphatase were added and further incubated for 2 h at 37 °C to yield free deoxynucleosides. To determine the amount of dG, hydrolyzed samples and 2′-deoxyguanosine (dG) and 8-OH-dG standards were injected onto a LiChrochart 250-4 (100RP18, 5 μm) HPLC column (Merck). Deoxyguanosine was detected at 255 nm, and calibration of the system was done with dG and 8-OH-dG as appropriate standards.Production of 11,12-Epoxyeicosatrienoic Acid (EET)—We used an HPLC electrospray MS2 method to detect 11,12-EET production in cultures of HCAECs as described (9Thum T. Borlak J. Circ. Res. 2004; 94: 1-13Crossref PubMed Google Scholar). HCAECs were treated with 100 μg/ml oxLDL for 24 h, and then production of 11,12-EET was analyzed.Intracellular ROS Measurements—After treatment with oxLDL (10, 20, and 100 μg/ml; 24 h) or native LDL (100 μg/ml; 24 h), cells were incubated with 10 μmol of 2′-7′-dichlorofluorescin diacetate (DCFH-DA) for 30 min at 37 °C. The 100 μg/ml oxLDL group was also concomitantly treated with the competitive oxLDL-receptor antagonist κ-carrageenan (250 μmol, 24 h). Endothelial cells were harvested, centrifuged for 5 min, and 1200 rpm at 4 °C, washed in PBS, and the resulting cell pellet was resuspended with 800 μl of PBS buffer. Fluorescence emission was detected at 530 nm ± 30 nm (fluorescin) after excitation of cells at 488 nm using flow cytometry (FACScan, Beckton Dickinson, Heidelberg, Germany).Preparation of Nuclear Extracts—HCAEC nuclear extracts were prepared by the modified Dignam C method (39Niehof M. Streetz K. Rakemann T. Bischoff S.C. Manns M.P. Horn F. Trautwein C. J. Biol. Chem. 2001; 276: 9016-9027Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). 24 h after treatment with oxLDL (100 μg/ml), oxLDL (100 μg/ml) + κ-carrageenan (250 μmol), oxLDL (100 μg/ml) + chelerythrine (1 μmol), or nLDL (100 μg/ml), cells were washed twice with ice-cold PBS, scraped into microcentrifuge tubes, and centrifuged for 5 min at 1780 × g, and 4 °C. Cell pellets were resuspended in hypotonic buffer (10 mmol of Tris, pH 7.4, 2 mmol of MgCl2, 140 mmol of NaCl, 1 mmol of dithiothreitol, 4 mmol of Pefabloc, 40 mmol of β-glycerophosphate, 1 mmol of Na3VO4, 10 μl of aprotinin/ml buffer, and 0.5% Triton X-100) for 10 min at 4 °C, transferred onto one volume of 50% sucrose in hypotonic buffer (see above), and centrifuged at 14,000 × g and 4 °C for 10 min. Nuclei were resuspended in Dignam C buffer (20 mmol of Hepes, pH 7.9, 25% glycerol, 420 mmol of NaCl, 1.5 mmol of MgCl2, 0.2 mmol of EDTA, 1 mmol of dithiothreitol, 4 mmol of Pefabloc, 40 mmol of β-glycerophosphate, 1 mmol of Na3VO4, and 10 μl of aprotinin/ml buffer) and gently rocked for 30 min at 4 °C. Nuclear debris was removed by centrifugation at 14,000 × g and 4 °C for 10 min, and extracts were aliquoted and stored at -80 °C. Protein concentrations were determined as described (40Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18442) Google Scholar).Western Blotting Experiments—Western immunoblotting was done as follows: Nuclear protein (100 μg) extracts from cultured endothelial cells were denaturated at 95 °C for 5 min, followed by SDS-PAGE on 12% polyacrylamide gels and blotted onto a polyvinylidene difluoride membrane (NEN, Dreieich, Germany) at 350 mA for 2 h in a buffer containing 400 mmol of glycine, 50 mmol of Tris, pH 8.3. Nonspecific binding sites were blocked with Rotiblock and 5% skim milk powder (Roth, Germany) in TBS buffer. After electroblotting of proteins, membranes were incubated with a polyclonal antibody for Oct-1 (dilution 1:50; sc-8024, Santa Cruz Biotechnology) for 1 h. For detection of human or murine LOX-1 expression we used a polyclonal LOX-1 antibody (ab60178; dilution 1:1000; Abcam, Cambridge, UK). After washing with TBS buffer containing 0.1% Tween-20. Subsequently, the membranes were incubated with a 1:5000 diluted anti-rabbit antibody (both for Oct-1 and LOX-1) for 1 h at room temperature followed by three successive washes with TBS buffer containing 0.1% Tween-20. Immunoreactive proteins were visualized with a chemiluminescence reagent kit (PerkinElmer Life Sciences, Dreieich, Germany) according to the manufacturer's instructions, and bands were scanned with the Kodak Image Station CF 440 and analyzed using the Kodak 1D 3.5 imaging software (Eastman Kodak Company).Annealing of Synthetic Oligonucleotides and 32P Labeling—Oligonucleotide sequences can be obtained from supplemental Table S1 and the corresponding 5′,3′ and 3′,5′ sequences were annealed at a concentration of 19.2 pmol/μl in 200 mmol of Tris (pH 7.6), 100 mmol of MgCl2, and 500 mmol of NaCl at 80 °C for 10 min and then cooled slowly to room temperature overnight and were stored at 4 °C. Annealed oligonucleotides were diluted to 1:10 in Tris-EDTA (pH 8.0) buffer and labeled using [32P]ATP (250 μCi, 3.000 Ci/mmol) and T4 polynucleotide kinase. End-labeled probes were purified from nonincorporated [32P]ATP by a Microspin G-25 column and eluted in a 100-μl volume.Electromobility Gel Shift Assay (EMSA)—The procedure for EMSA was adapted from a previously described method (39Niehof M. Streetz K. Rakemann T. Bischoff S.C. Manns M.P. Horn F. Trautwein C. J. Biol. Chem. 2001; 276: 9016-9027Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Briefly, 5 μg of endothelial nuclear extract was incubated with binding buffer consisting of 25 mmol HEPES (pH 7.6), 5 mmol of MgCl2, 34 mmol of KCl, 2 mmol of dithiothreitol, 2 mmol of Pefabloc, 0.5 μl of aprotinin (2.2 mg/ml), 50 ng of poly(dl-dC), and 80 ng of bovine serum albumin. The binding reaction was carried out for 20 min on ice and free DNA and DNA-protein complexes were resolved on a 6% polyacrylamide gel. Competition studies were done by adding a specified amount (50-fold) of unlabeled oligonucleotides or a specific Oct-1 antibody to the reaction mix 10 min before addition of the labeled oligonucleotides. In addition, we also incubated nuclear extracts with excess addition of mutated Oct-1 consensus binding sites. Gels were blotted to Whatman 3 MM paper, dried under vacuum, exposed to imaging screens (Imaging Screen-K, Bio-Rad) for autoradiography for 24 h at room temperature and analyzed using a phosphorimaging system (Molecular Imager FX pro plus; Bio-Rad) and the Quantity One Version 4.2.2 software (Bio-Rad).Immunohistochemistry—HCAECs were fixed and permeabilized as described (41Thum T. Hoeber S. Froese S. Klink I. Stichtenoth D.O. Galuppo P. Jakob M. Tsikas D. Anker S.D. Poole-Wilson P.A. Borlak J. Ertl G. Bauersachs J. Circ. Res. 2007; 100: 434-443Crossref PubMed Scopus (255) Google Scholar). Oct-1 expression was determined after addition of an anti-Oct-1 antibody (Abcam; ab51363; dilution 1:10) for 2 h at 37 °C. Thereafter, cells were washed and an Alexa Fluor 488 goat anti-mouse IgG was added for 1 h at 37 °C (dilution 1:200). In addition, nuclei were stained with 4′,6-diamidino-2-phenylindol (DAPI). Then cells were observed under appropriate fluorescence microscopy conditions as described (41Thum T. Hoeber S. Froese S. Klink I. Stichtenoth D.O. Galuppo P. Jakob M. Tsikas D. Anker S.D. Poole-Wilson P.A. Borlak J. Ertl G. Bauersachs J. Circ. Res. 2007; 100: 434-443Crossref PubMed Scopus (255) Google Scholar).Transcription Factor Binding Sites in Cytochrome P450 Promoters—We searched for Oct-1 binding sites in the promoters of CYP1A1, CYP2A6/7, CYP2B6, CYP2C8, CYP2C9, CYP2E1, CYP2J2, and eNOS using the transcription factor data base TRANSFAC Professional 6.2 and the program Matrix Search for Transcription Factor Binding Sites (MATCH™ 1.7; Biobase). Core and matrix similarities for all binding sites were set to 1.0 and 0.85, respectively, to obtain highly specific results.Small Interference RNA (siRNA)-mediated Knockdown of Oct-1 in HCAECs—HCAECs were cultured to 70–80% of confluence and were transfected with Oct-1-siRNA using the BLOCK-iT™ Transfection kit (Invitrogen). We incubated HCAECs with the siRNA oligonucleotide Oct-1 (50 nm; sc-36119, Santa Cruz Biotechnology) for 48 h to down-regulate Oct-1 expression. FITC-labeled scrambled siRNA (Control-FITC block-it fluorescent Oligo 2013, Invitrogen) was used as a negative and transfection control. Oct-1 expression was monitored by Western blotting (see above).RESULTSOxLDL Induced Oxidative DNA Damage in HCAECs—LOX-1 was highly expressed in atherosclerotic aortic tissue of ApoE-/- mice fed a high lipid diet or humans aortae of patients diagnosed with ischemic heart disease when compared with appropriate controls (Fig. 1A). This suggests pathophysiological relevance of LOX-1 up-regulation in atherosclerosis. We next focused on effects of LOX-1-mediated oxLDL uptake in HCAEcs in vitro. Based on microscopic evaluation, there were no signs of altered morphology or cellular toxicity after treatment of cell cultures with increasing doses of oxLDL (10–300 μg/ml; 24 h). LDH leakage was used as a marker for membrane integrity and was assayed after treatment of cell cultures with incremental doses of oxLDL (24 h). Notably, oxLDL induced LDH leakage into culture medium with >40 units/liter at the highest dose (data not shown). Based on LDH activity, we used 100 μg/ml oxLDL as the best tolerable non-cytotoxic dose. We chose the 24-h time point for subsequent studies, because we and others (9Thum T. Borlak J. Circ. Res. 2004; 94: 1-13Crossref PubMed Google Scholar, 42Hu B. Li D. Sawamura T. Mehta J.L. Biochem. Biophys. Res. Commun. 2003; 307: 1008-1012Crossref PubMed Scopus (19) Google Scholar) demonstrated enhanced ROS production in HCAECs to be significantly elevated after 24 h of treatment with oxLDL. The expression of PECAM-1 (CD31) served as an endothelial differentiation marker and was expressed by >95% of cultured cells, as determined by flow cytometry (data not shown). Treatment of HCAECs with oxLDL (100 μg/ml; 24 h) increased intracellular ROS production by 4.5-fold, whereas native (non-oxidized) LDL had no effect (Fig. 1B). ROS production was attenuated when κ-carrageenan, a competitive LOX-1 inhibitor, was added (Fig. 1B). Treatment of HCAECs with oxLDL resulted in a dose-dependent 4-fold increase of the stress-inducible transcripts GADD45 and GADD153 (Fig. 2A). Both GADDs were additionally up-regulated in atherosclerotic human aortae (Fig. 2B). Likewise, moxLDL increased expression of GADDs (Fig. 3A). Production of 8-OH-dG was up to 3-fold increased after treatment of HCAECs with oxLDL (Fig. 2C). LOX-1 inhibition with κ-carrageenan abrogated induction of GADD transcripts and formation of the 8-OH-dG DNA adduct (Fig. 2). Addition of native LDL (100 μg/ml) had no effect.FIGURE 2A, semiquantitative gene expression of GADD45 and GADD153 in control, nLDL (100 μg/ml), and oxLDL (10–100 μg/ml)-treated endothelial c" @default.
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• W2003753810 date "2008-07-01" @default.
• W2003753810 modified "2023-10-09" @default.
• W2003753810 title "LOX-1 Receptor Blockade Abrogates oxLDL-induced Oxidative DNA Damage and Prevents Activation of the Transcriptional Repressor Oct-1 in Human Coronary Arterial Endothelium" @default.
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• W2003753810 doi "https://doi.org/10.1074/jbc.m708309200" @default.
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