Oxidative Stress and Compartment of Gene Expression Determine Proatherosclerotic Effects of Inducible Nitric Oxide Synthase
2009; Elsevier BV; Volume: 174; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080730
ISSN1525-2191
AutoresPadmapriya Ponnuswamy, Eva Ostermeier, Angelika Schröttle, Jiqiu Chen, Paul L. Huang, Georg Ertl, Bernhard Nieswandt, Peter Kuhlencordt,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoGenetic and pharmacological inhibition of inducible nitric oxide synthase (iNOS) decreases atherosclerosis development. Potential proatherogenic effects of iNOS include iNOS mediated oxidative stress and iNOS expression in different cellular compartments. Lesional iNOS can potentially produce nitric oxide radicals (NO), superoxide radicals (O2−), or both; these radicals may then react to form peroxynitrite. Alternatively, O2− radicals from oxidases co-expressed with iNOS could react with NO to produce peroxynitrite. Therefore, the expression profiles of the genes that modulate the redox system in different iNOS-expressing cell compartments may determine the role of iNOS in atherosclerosis. We used apoE (apoE−/−) and apoE/iNOS double knockout (apoE−/−/ iNOS−/−) mice to assess vascular NO, O2−, and peroxynitrite formation by electron spin resonance spectroscopy, high performance liquid chromatography, and 3-nitrotyrosine staining. The relevance of the iNOS expressing cell compartment was tested by bone marrow transplantation. We show that iNOS significantly contributes to vascular NO production and itself produces O2−, leading to peroxynitrite formation in atherosclerotic lesions. Our bone marrow transplantation experiments show that bone marrow derived cells exclusively mediate the proatherosclerotic effects of iNOS in males, while both parenchymal and bone marrow derived iNOS equally contribute to atherosclerosis in females. Moreover, iNOS expression affects vascular remodeling. These findings establish for the first time that the proatherosclerotic effects of iNOS vary with sex in addition to the compartment of its expression. Genetic and pharmacological inhibition of inducible nitric oxide synthase (iNOS) decreases atherosclerosis development. Potential proatherogenic effects of iNOS include iNOS mediated oxidative stress and iNOS expression in different cellular compartments. Lesional iNOS can potentially produce nitric oxide radicals (NO), superoxide radicals (O2−), or both; these radicals may then react to form peroxynitrite. Alternatively, O2− radicals from oxidases co-expressed with iNOS could react with NO to produce peroxynitrite. Therefore, the expression profiles of the genes that modulate the redox system in different iNOS-expressing cell compartments may determine the role of iNOS in atherosclerosis. We used apoE (apoE−/−) and apoE/iNOS double knockout (apoE−/−/ iNOS−/−) mice to assess vascular NO, O2−, and peroxynitrite formation by electron spin resonance spectroscopy, high performance liquid chromatography, and 3-nitrotyrosine staining. The relevance of the iNOS expressing cell compartment was tested by bone marrow transplantation. We show that iNOS significantly contributes to vascular NO production and itself produces O2−, leading to peroxynitrite formation in atherosclerotic lesions. Our bone marrow transplantation experiments show that bone marrow derived cells exclusively mediate the proatherosclerotic effects of iNOS in males, while both parenchymal and bone marrow derived iNOS equally contribute to atherosclerosis in females. Moreover, iNOS expression affects vascular remodeling. These findings establish for the first time that the proatherosclerotic effects of iNOS vary with sex in addition to the compartment of its expression. Atherosclerosis is considered a chronic inflammatory disease.1Ross R Atherosclerosis–an inflammatory disease.N Engl J Med. 1999; 340: 115-126Crossref PubMed Scopus (19278) Google Scholar The cellular and molecular mechanisms that initiate and propagate atherosclerosis are complex and involve the expression of inducible nitric oxide synthase (iNOS) in early and advanced atherosclerotic plaques.2Wilcox JN Subramanian RR Sundell CL Tracey WR Pollock JS Harrison DG Marsden PA Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels.Arterioscler Thromb Vasc Biol. 1997; 17: 2479-2488Crossref PubMed Scopus (420) Google Scholar In healthy vessels iNOS is usually not expressed. However, in response to stimulation with inflammatory cytokines/bacterial by-products or low pH (pH 7.0) in the microenvironment of inflammatory lesions, a wide range of tissues and cell types can express iNOS.3MacMicking J Xie QW Nathan C Nitric oxide and macrophage function.Annu Rev Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3483) Google Scholar, 4Bellocq A Suberville S Philippe C Bertrand F Perez J Fouqueray B Cherqui G Baud L Low environmental pH is responsible for the induction of nitric-oxide synthase in macrophages. Evidence for involvement of nuclear factor-kappaB activation.J Biol Chem. 1998; 273: 5086-5092Crossref PubMed Scopus (183) Google Scholar Aside from its transcriptional regulation, multiple post-translational modifications of iNOS have been identified that may allow complex regulations of the catalytic activity of iNOS.5Giordano A Tonello C Bulbarelli A Cozzi V Cinti S Carruba MO Nisoli E Evidence for a functional nitric oxide synthase system in brown adipocyte nucleus.FEBS Lett. 2002; 514: 135-140Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Kolodziejska KE Burns AR Moore RH Stenoien DL Eissa NT Regulation of inducible nitric oxide synthase by aggresome formation.Proc Natl Acad Sci USA. 2005; 102: 4854-4859Crossref PubMed Scopus (57) Google Scholar, 7Navarro-Lerida I Corvi MM Barrientos AA Gavilanes F Berthiaume LG Rodriguez-Crespo I Palmitoylation of inducible nitric-oxide synthase at Cys-3 is required for proper intracellular traffic and nitric oxide synthesis.J Biol Chem. 2004; 279: 55682-55689Crossref PubMed Scopus (37) Google Scholar, 8Pan J Burgher KL Szczepanik AM Ringheim GE Tyrosine phosphorylation of inducible nitric oxide synthase: implications for potential post-translational regulation.Biochem J. 1996; 314: 889-894Crossref PubMed Scopus (84) Google Scholar, 9Saini R Patel S Saluja R Sahasrabuddhe AA Singh MP Habib S Bajpai VK Dikshit M Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies.J Leukoc Biol. 2006; 79: 519-528Crossref PubMed Scopus (64) Google Scholar iNOS protein produces high amounts of nitric oxide (NO), which is highly reactive with other free radicals. NO reacts with superoxide (O2−) to form peroxynitrite, which in turn leads to protein nitration, DNA damage, and poly (ADP-ribose) polymerase activation.10Beckman JS Beckman TW Chen J Marshall PA Freeman BA Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.Proc Natl Acad Sci USA. 1990; 87: 1620-1624Crossref PubMed Scopus (6718) Google Scholar Peroxynitrite reacts with other molecules to form a variety of oxygen and nitrogen free radicals ie, nitrogen dioxide, peroxynitrous acid, and hydroxyl radical.11Hall ED Detloff MR Johnson K Kupina NC Peroxynitrite-mediated protein nitration and lipid peroxidation in a mouse model of traumatic brain injury.J Neurotrauma. 2004; 21: 9-20Crossref PubMed Scopus (195) Google Scholar Therefore, the predominant role of iNOS is thought to relate to oxidative stress mediated host defense against tumor cells and microorganisms.3MacMicking J Xie QW Nathan C Nitric oxide and macrophage function.Annu Rev Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3483) Google Scholar However, iNOS can also act as an anti-inflammatory molecule. For example, iNOS−/− mice showed an impaired ability to resolve colonic inflammation and increased neutrophil infiltration in a model of acetic acid induced acute colitis12McCafferty DM Mudgett JS Swain MG Kubes P Inducible nitric oxide synthase plays a critical role in resolving intestinal inflammation.Gastroenterology. 1997; 112: 1022-1027Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar and lipopolysaccharide-treated iNOS−/− mice show increased neutrophil endothelial interactions in post capillary venules of the cremaster muscle assessed by intravital microscopy.13Hickey MJ Sharkey KA Sihota EG Reinhardt PH Macmicking JD Nathan C Kubes P Inducible nitric oxide synthase-deficient mice have enhanced leukocyte-endothelium interactions in endotoxemia.FASEB J. 1997; 11: 955-964Crossref PubMed Scopus (251) Google Scholar Whether iNOS also influences leukocyte endothelial-interactions in atherosclerosis, where mononuclear cells are the predominant subset of leukocytes interacting with the endothelium of large arteries is currently unknown. Several reasons may explain the beneficial or detrimental effects of iNOS. The role of iNOS may depend on the phase of inflammation. For example, studies of experimental traumatic brain injury suggest that early after injury, iNOS may be detrimental via peroxynitrite formation while long term iNOS expression following brain injury seems to be protective via antioxidant effects.14Bayir H Kagan VE Borisenko GG Tyurina YY Janesko KL Vagni VA Billiar TR Williams DL Kochanek PM Enhanced oxidative stress in iNOS-deficient mice after traumatic brain injury: support for a neuroprotective role of iNOS.J Cereb Blood Flow Metab. 2005; 25: 673-684Crossref PubMed Scopus (104) Google Scholar The antioxidant effects of NO are explained by its ability to scavenge oxygen free radicals and to inhibit lipid peroxidation. It has been suggested that the balance between local concentrations of NO and O2− may determine whether peroxynitrite forms and lipid peroxidation occurs.15Darley-Usmar V Wiseman H Halliwell B Nitric oxide and oxygen radicals: a question of balance.FEBS Lett. 1995; 369: 131-135Abstract Full Text PDF PubMed Scopus (524) Google Scholar Conditions that favor peroxynitrite formation would favor lipid peroxidation, whereas excess NO without O2− may block lipid peroxidation by scavenging peroxyl radicals. In a model of chronic atherosclerosis, the apoE−/− mouse, iNOS deficiency potently reduces plaque development on a high fat diet, suggesting that iNOS is proatherogenic.16Detmers PA Hernandez M Mudgett J Hassing H Burton C Mundt S Chun S Fletcher D Card DJ Lisnock J Weikel R Bergstrom JD Shevell DE Hermanowski-Vosatka A Sparrow CP Chao YS Rader DJ Wright SD Pure E Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice.J Immunol. 2000; 165: 3430-3435PubMed Scopus (185) Google Scholar, 17Kuhlencordt PJ Chen J Han F Astern J Huang PL Genetic deficiency of inducible nitric oxide synthase reduces atherosclerosis and lowers plasma lipid peroxides in apolipoprotein E-knockout mice.Circulation. 2001; 103: 3099-3104Crossref PubMed Scopus (220) Google Scholar Moreover, chronic treatment of hypercholesterolemic rabbits with a pharmacological iNOS inhibitor decreases atherosclerosis development18Hayashi T Matsui-Hirai H Fukatsu A Sumi D Kano-Hayashi H Rani PJ Iguchi A Selective iNOS inhibitor. ONO1714 successfully retards the development of high-cholesterol diet induced atherosclerosis by novel mechanism.Atherosclerosis. 2006; 187: 316-324Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 19Behr-Roussel D Rupin A Simonet S Bonhomme E Coumailleau S Cordi A Serkiz B Fabiani JN Verbeuren TJ Effect of chronic treatment with the inducible nitric oxide synthase inhibitor N-iminoethyl-l-lysine or with l-arginine on progression of coronary and aortic atherosclerosis in hypercholesterolemic rabbits.Circulation. 2000; 102: 1033-1038Crossref PubMed Scopus (82) Google Scholar and gene transfer mediated increased vascular iNOS expression results in impaired NO dependent vasodilation.20Xia Y Zweier JL Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages.Proc Natl Acad Sci USA. 1997; 94: 6954-6958Crossref PubMed Scopus (599) Google Scholar, 21Gunnett CA Lund DD Chu Y Brooks 2nd, RM Faraci FM Heistad DD NO-dependent vasorelaxation is impaired after gene transfer of inducible NO-synthase.Arterioscler Thromb Vasc Biol. 2001; 21: 1281-1287Crossref PubMed Scopus (54) Google Scholar In contrast, transient gene transfer mediated expression of iNOS decreases smooth muscle cell proliferation and prevents neointima formation following balloon angioplasty (vascular injury model) in rats and pigs.22Shears 2nd, LL Kibbe MR Murdock AD Billiar TR Lizonova A Kovesdi I Watkins SC Tzeng E Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo.J Am Coll Surg. 1998; 187: 295-306Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar Addition of NO donors, as well as iNOS mediated NO production in vascular smooth muscle cells, inhibits cell proliferation.23Garg UC Hassid A Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells.J Clin Invest. 1989; 83: 1774-1777Crossref PubMed Scopus (1994) Google Scholar In mice, iNOS protects from developing transplant arteriosclerosis by inhibiting neointimal smooth muscle accumulation.24Koglin J Glysing-Jensen T Mudgett JS Russell ME Exacerbated transplant arteriosclerosis in inducible nitric oxide-deficient mice.Circulation. 1998; 97: 2059-2065Crossref PubMed Scopus (114) Google Scholar The seemingly opposing results may be due to differences in the cellular composition of the vessel wall and the compartment of iNOS expression in chronic atherosclerosis versus transplant arteriosclerosis or smooth muscle cell proliferation following balloon angioplasty. iNOS expression relevant to atherosclerosis development was detected in vascular smooth muscle cells, mononuclear cells, and lymphocytes. These various cellular sources are capable of generating different amounts of iNOS and subsequently target iNOS expression to various compartments of the plaque. Moreover, the cellular source determines a specific array of genes co-expressed with iNOS, which may influence their redox status.25Kubes P Inducible nitric oxide synthase: a little bit of good in all of us.Gut. 2000; 47: 6-9Crossref PubMed Scopus (98) Google Scholar For example, leukocytes can produce substantial amounts of NO and O2− from iNOS and NADPH oxidase, resulting in the formation of peroxynitrite.10Beckman JS Beckman TW Chen J Marshall PA Freeman BA Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.Proc Natl Acad Sci USA. 1990; 87: 1620-1624Crossref PubMed Scopus (6718) Google Scholar Peroxynitrite can oxidize low density lipoprotein and cause nitrosylation of proteins, which influences protein function.26Beckmann JS Ye YZ Anderson PG Chen J Accavitti MA Tarpey MM White CR Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry.Biol Chem Hoppe Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1071) Google Scholar Moreover, under conditions of substrate (l-arginine) or cofactor (tetrahydrobiopterin) deficiency NOS can "uncouple" to generate O2− instead of NO.27Xia Y Roman LJ Masters BS Zweier JL Inducible nitric-oxide synthase generates superoxide from the reductase domain.J Biol Chem. 1998; 273: 22635-22639Crossref PubMed Scopus (349) Google Scholar Even more intriguing iNOS is likely capable of producing NO and O2− simultaneously, which could directly lead to peroxynitrite formation.20Xia Y Zweier JL Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages.Proc Natl Acad Sci USA. 1997; 94: 6954-6958Crossref PubMed Scopus (599) Google Scholar Whether iNOS is "uncoupled" in atherosclerosis and produces substantial amounts of O2− in addition to NO is currently unknown. To design treatment strategies that decrease iNOS expression in chronic atherosclerosis without interfering with potentially beneficial effects of iNOS, ie, prevention of neointima proliferation following balloon angioplasty or host defense against infectious or neoplastic disease, a detailed understanding of the role of iNOS in plaque development is required. Therefore, the current study was designed to assess possible mechanisms involved in the proatherogenic effects of iNOS. We used apoE−/− and apoE−/−/iNOS−/− mice, which allowed testing the effects of iNOS deficiency on spontaneous atherosclerosis development. Since theoretically iNOS is capable of producing both NO and O2−, we investigated the contribution of iNOS to vascular NO production by electron spin resonance (ESR) spectroscopy, oxidative stress by ESR and high performance liquid chromatography (HPLC) measurements of O2− and nitrosative stress by quantification of 3-nitrotyrosine. ESR and HPLC methods provide direct detection of NO and O2− production with utmost specificity and sensitivity. To assess the role of the iNOS expressing cellular compartment in atherosclerosis we studied plaque formation following bone marrow transplantation (BMT) between apoE−/− and apoE−/−/iNOS−/− animals. Using this approach, we investigated atherosclerosis development in animals selectively expressing iNOS in bone marrow derived versus parenchymal cells of the vessel wall. All procedures performed conformed with the policies of the University of Würzburg, the NIH guidelines and an independent governmental committee for care and use of laboratory animals. ApoE−/− and iNOS−/− animals were obtained from The Jackson Laboratories. All mice were backcrossed for ten generations to the C57Bl6 strain. Ly5.2/Cr mice on a C57Bl6 genetic background, carrying a mutation of their common leukocyte antigen (designated CD45.1) were obtained from the National Cancer Institute. iNOS−/− and apoE−/− animals were crossed to generate double knockout apoE−/−/iNOS−/− mice. Mice were genotyped by PCR using protocols supplied by The Jackson Laboratories. ApoE−/−/iNOS−/− or apoE−/−/iNOS+/+ were used as bone marrow donors or recipients to generate the following two "chimeric" mice strains. Bone marrow cells of apoE−/−/iNOS+/+ were transplanted into apoE−/−/ iNOS−/− mice to generate mice that lack parenchymal iNOS but express iNOS in blood cells (apoE−/−/iNOS+/+ in apoE−/−/iNOS−/−). Additionally, bone marrow cells of apoE−/−/iNOS−/− were transplanted into apoE−/−/iNOS+/+ mice to generate mice expressing iNOS in parenchymal cells, but lack iNOS in circulating blood cells (apoE−/−/iNOS−/− in apoE−/−/iNOS+/+). The animals were 10 weeks old when used for BMT experiments. As controls, apoE−/−/iNOS−/− received apoE−/−/iNOS−/− marrow, generating mice that lack iNOS completely. Moreover, apoE−/−/iNOS+/+ mice were transplanted with apoE−/−/iNOS+/+ bone marrow cells to generate controls expressing iNOS in both compartments (Table 1). A second line of mice was generated crossing apoE−/− with Ly5.2/Cr (Ly5.1+/+) animals. These mice were genotyped for apoE by PCR and for the Ly5.1 locus by fluorescence-activated cell sorting analysis of peripheral blood cells. ApoE−/−/Ly5.1+/+ mice were used as donors to evaluate transplantation efficiency. Four weeks after BMT a high fat "western-type" diet (Harlan Teklad, Madison, Wisconsin) was started and maintained for 16 weeks. Animals used in experiments that did not involve BMT were weaned at 3 weeks of age and fed with "western-type" diet for 18 weeks.Table 1Characteristics of BMT AnimalsBody weightTotal area aorta (mm2) maleTotal area aorta (mm2) femaleTotal cholesterol (mg/dl)GenotypennnnC57Bl628 ± 0.51278 ± 1.412apoE−/−25 ± 0.62377 ± 2.91067 ± 1.216940 ± 5522apoE−/− in apoE−/−/iNOS−/−27 ± 0.91574 ± 2.4670 ± 1.511994 ± 5318apoE−/−/iNOS−/−in apoE−/−26 ± 0.92067 ± 1.3*P < 0.05 vs. all groups; n, number of animals analyzed.860 ± 1.1*P < 0.05 vs. all groups; n, number of animals analyzed.81187 ± 8416apoE−/−/iNOS−/−25 ± 0.71379 ± 2.1876 ± 2.35927 ± 13910* P < 0.05 vs. all groups; n, number of animals analyzed. Open table in a new tab NO production of aortic rings was measured in an organ bath using colloid iron (II) diethyldithiocarbamate (Fe[DETC]2) as a spin trap and ESR detection (e-scan ESR spectrometer, Bruker BioSpin GmbH, Karlsruhe, Baden Wurttemburg). The method was adapted for detection of baseline NO production in apoE−/− mice.28Kleschyov AL Munzel T Advanced spin trapping of vascular nitric oxide using colloid iron diethyldithiocarbamate.Methods Enzymol. 2002; 359: 42-51Crossref PubMed Scopus (52) Google Scholar Briefly, animals were anesthetized with Avertin (80 μg/kg i.p.). The aorta was removed quickly and cleaned off adherent tissue at 4°C in chilled Krebs-Hepes Buffer (KHB) using a cold plate (Noxygen Science Transfer & Diagnostics, Denzlingen, Germany). The aortas were cut into 2 mm rings and were placed in one well of a 12-well plate containing 1500 μl KHB. The aortic NO produced was trapped with Fe-(DETC)2 for 1 hour in 37°C KHB. The protein concentration of the samples were quantified with a BCA protein assay kit (Pierce, Rockford, IL) and used to normalize the ESR signal intensity. The instrumental settings are mentioned in Table 2.Table 2Instrumental Settings Used for ESR MeasurementsInstrumental settingExperimentVascular NOVascular superoxideCentre field3308 G3388 GSweep width80 G132 GMicrowave frequency9.495 GHz9.497 GHzMicrowave power50 mW1.25 mWModulation amplitude4.6 G1.63 GModulation frequency86 kHz86 kHzTime constant81.92 ms40.96 msConversion time20.48 ms10.24 msNumber of scans10050 Open table in a new tab Production of O2− was measured in aortic rings in an organ bath in 37°C KHB. The experimental procedure was performed according to a previously published protocol29Dikalova A Clempus R Lassegue B Cheng G McCoy J Dikalov S San Martin A Lyle A Weber DS Weiss D Taylor WR Schmidt HH Owens GK Lambeth JD Griendling KK Nox1 overexpression potentiates angiotensin II-induced hypertension and vascular smooth muscle hypertrophy in transgenic mice.Circulation. 2005; 112: 2668-2676Crossref PubMed Scopus (358) Google Scholar using the above mentioned e-scan spectroscope. Instrumental settings are reported in Table 2. O2− production was assessed by pre incubating aortic rings with PEG-SOD (100 U/ml) parallel to 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine for 1 hour. The intensity of the ESR signal was normalized to the sample's protein content. HPLC measurements served as a second, independent method for O2− detection. O2− was measured in aortic rings by detection of oxyethidium, the fluorescent reaction product of O2− and dihydroethidium.30Zhao H Kalivendi S Zhang H Joseph J Nithipatikom K Vasquez-Vivar J Kalyanaraman B Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide.Free Radic Biol Med. 2003; 34: 1359-1368Crossref PubMed Scopus (651) Google Scholar Briefly, vessel rings were incubated in an organ bath with KHB containing 50 μmol/L dihydroethidium at 37°C. Subsequently, dihydroethidium was washed off and the rings were incubated for 1 hour. Aortic rings were then homogenized in ice cold methanol. The homogenate was filtered and separated by reverse phase HPLC using a C-18 column (Nucleosil 250, 4.5 mm; Sigma-Aldrich, Munich, Bayern) on a AKTA HPLC system (Amersham Biosciences, GE Health care, Munich, Bayern). Oxyethidium was quantified with a fluorescence detector (Jasco, Great Dunmov, Essex). The detected oxyethidium was normalized to the sample's protein content.31Fink B Laude K McCann L Doughan A Harrison DG Dikalov S Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay.Am J Physiol Cell Physiol. 2004; 287: C895-C902Crossref PubMed Scopus (214) Google Scholar Recipient animals were housed in autoclaved, single ventilated microisolator-cages. Animals were treated with acidified water (pH 2.0) containing 100 mg/L neomycin and 10 mg/ml polymyxin B sulfate (Sigma Aldrich) for ten days prior, until 2 weeks after lethal irradiation. Mice were irradiated with 1000 rads from a cesium γ source, followed by BMT 4 hours later.32Linton MF Atkinson JB Fazio S Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation.Science. 1995; 267: 1034-1037Crossref PubMed Scopus (410) Google Scholar Bone marrow cells were prepared from the tibia and femur bones by flushing the bones with RPMI 1640 media (Gibco, Grand Island, NY) containing 2% fetal bovine serum and 400 U/ml DNase. Cells were filtered through a spleen mesh, counted and resuspended in media 199 containing 1% Hepes buffer, 400 U/ml DNase and 200 μg/ml gentamicin. Recipient animals received ∼5 × 106 bone marrow cells in 0.5 ml of BMT medium (formula) by tail vein injection. To evaluate transplantation efficiency, apoE−/− animals were transplanted with ∼5 × 106 bone marrow cells from either apoE−/−/Ly5.2 or apoE−/−/Ly5.1, following the above protocol. Two weeks following BMT, animals were anesthetized with isofluorane and a 100 μl blood sample was drawn from the retro-orbital venous plexus. The red blood cells were subsequently lysed using red cell lysis buffer (BD Biosciences, Heidelberg, Baden Wurttemburg). The white cell pellet was resuspended in PBS and 1% fetal bovine serum, and stained with a monoclonal PE-anti-mouse CD45.1 and a fluorescence isothiocyanate-anti-mouse CD45.2 antibodies (BD Biosciences). The fluorescence of 5 × 104 cells was measured on a FACScan (BD Biosciences). The aorta was dissected and analyzed as previously described.33Kuhlencordt PJ Gyurko R Han F Scherrer-Crosbie M Aretz TH Hajjar R Picard MH Huang PL Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice.Circulation. 2001; 104: 448-454Crossref PubMed Scopus (517) Google Scholar Briefly, animals were anesthetized with Avertin (80 μg/kg i.p.), the aorta was perfused with PBS, pH 7.4, dissected from the aortic valve to the iliac bifurcation and fixed in 4% paraformaldehyde. Adventitial tissue was removed and the aorta was opened longitudinally and pinned onto a black wax surface using micro needles (Fine Science Tools, Heidelberg, Baden Wurttemburg). Serial images of the submerged vessels were captured with a black and white video camera (COHU, Poway, CA) mounted on the c-mount of a stereomicroscope (Leica, Wetzlar, Hessen). Lipid rich intraluminal lesions were subsequently stained with Sudan IV. Serial color pictures were captured using the same microscope equipped with a Leica 35 mm camera and used to identify lesions. Image analysis of digital images was performed using Image Pro Plus (Version 4.1; Media Cybernetics, Bethesda, MD). The amount of aortic lesion formation in each animal was measured as percent lesion area per total area of the aorta. Aortic arches were embedded in Tissue-Tek (Sakura Finetek, Heppenheim, Hesse) and snap-frozen in liquid nitrogen. Serial sections were fixed in acetone before staining. Immunostaining was performed using an anti-nitrotyrosine antibody (Upstate Biotechnology Inc., Lake Placid, NY). Biotinylated anti-rabbit IgG (Vector Laboratoties, Burlingame, CA) was used as the secondary antibody, and the nitrotyrosine staining was visualized using Vectastain ABC kit and DAB as the substrate (Vector laboratories). Photomicrographs of the vessel sections were taken with a Leitz-camera mounted on a light microscope (Carl-Zeiss, Munich, Bayern). Pictures were digitalized and transferred to a computer for planimetry using Image Pro Plus (Media Cybernetics). All images were analyzed at 400-fold magnification. Results were expressed as positive staining area per total area of the plaque. Aortic protein was isolated using RIPA buffer and western blots were performed using a polyclonal anti-iNOS antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). To confirm equal loading of protein an anti-α-actin-antibody (Santa Cruz Biotechnology, Inc.) was used. Blots were analyzed densitometrically using Quantity One software (Bio-Rad Laboratories, Munich, Bayern) and the density of protein bands for iNOS were normalized to the corresponding α-actin bands of the same blots. First-strand cDNA was synthesized from 2 μg of total aortic RNA, using random primers (Fermentas, St. Leon-Rot, Baden Wurttemburg). mRNA expression of nNOS and eNOS was quantified by real-time PCR (iCycler, Bio-Rad Laboratories). PCR amplification was performed for 40 cycles at primer annealing of 60°C. The expression of nNOS and eNOS was normalized to HPRT signal. The primer pairs used for real-time PCR are shown in Table 3.Table 3Sequences of Primers and Probes Used for Real-Time PCRGenePrimers and Flurogenic probesnNOSSense: 5′-CCCACCAAAGCTGTCGATCT-3′Antisense: 5′-GGAGGTTGGCCTTGGTATTT-3′5′-6FAM-CACACCATTAGCCTGGGAGACTGAGCC-TMReNOSSense: 5′-CTGGCAGCCCCAAGACCTA-3′Antisense: 5′-CGATGACGTCACCGGCTT-3′5′-6FAM-TCCTGAGGACAGAGCTAGCCGCGGAXT-PhosphateHPRTSense: 5′-GTTGGATACAGGCCAGACTTTGT-3′Antisense: 5′-CCACAGGACTAGAACACCTGC-3′5′-6FAM-CTCGTATTTGCAGATTCAACTTGCGCXT-PH Open table in a new tab Lipoprotein cholesterol distribution of plasma samples was evaluated after fractionation by fast protein liquid chromatography gel filtration. Separate plasma samples (200 μl) from four animals per sex and genotype were fractionated on two serial superose-6-columns using an AKTA-BASIC system. Total cholesterol of plasma samples and fast protein liquid chromatography-fractions were measured using Infinity Cholesterol Reagent (Thermo Electron, Melbourne, Victoria) and a Spectra MAX 250 photometer (Molecular Devices, Sunnyvale, CA). Statistical analyses were performed using Stat View 4.51 (Abacus Concepts, Inc., Berkeley, CA) and Sigma Plot (Version 8, Systat Software Inc., San Jose, CA). Data were analyzed using Student's t-test or two way analysis of variance, followed by S
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