A Common Polymorphism in EC-SOD Affects Cardiopulmonary Disease Risk by Altering Protein Distribution John M. Hartney, Timothy Stidham, David A. Goldstrohm, Rebecca E. Oberley-Deegan, Michael R. Weaver, Zuzana Valnickova-Hansen, Carsten Scavenius, Richard K.P. Benninger, Katelyn F. Leahy, Richard Johnson, Fabienne Gally, Beata Kosmider, Angela K. Zimmermann, Jan J. Enghild, Eva Nozik-Grayck and Russell P. Bowler Circ Cardiovasc Genet. published online August 1, 2014; Circulation: Cardiovascular Genetics is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 1942-325X. Online ISSN: 1942-3268
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DOI: 10.1161/CIRCGENETICS.113.000504
A Common Polymorphism in EC-SOD Affects Cardiopulmonary Disease Risk by Altering Protein Distribution
Running title: Hartney et al.; EC-SOD polymorphism alters distribution
John M. Hartney, PhD1,2*; Timothy Stidham, MD3*; David A. Goldstrohm, PhD1*; Rebecca E. Oberley-Deegan, PhD1; Michael R. Weaver, BS1; Zuzana Valnickova-Hansen, BSc4; Carsten Scavenius, PhD4; Richard K.P. Benninger, PhD3,5; Katelyn n F. L Leahy, eahy ea hy, BS1; Ri Rich Richard ch Johnson, BS3; Fabienne Gally, PhD1; Beata Kosmider, PhD1; Angelaa K. Zi Zimmermann Zimmermann, n, P PhD6; n J. Eng Enghild nghi gh ld d, Ph PhD hD4; Eva Nozik-Grayck, Nozik Grayck MD3*; Russell P. P Bowler, Bowler B o ler, MD, ow MD PhD1* Jan Enghild,
1
Department n of nt of Medicine, M dicine Me ne, Na ne National ation on nal Jew Jewish ewish He H Health; altth; 2In al Integrated ntegr grated ed Department Dep epartm tment off Immunology, Imm mm munol olog ol ogy, og y,, Un University nivverr of 3 5 Colorado Denver National Jewish Health, Denver; d De do Denv nv ver aand nd N atio at iona io nall Jewi na wish wi shh H ealt lthh, D lt enve verr; De ve Department Dep part rtme rt mentt of ment of Pediatrics, Peedi d at atri rics ri cs, De cs Department Depa part pa rtme rt ment me nt of e eering, Universi ity of of Colorado Colo Co lo ora rado do School Sch hoo o l of Medicine, Med edic iccin ine, e Aurora, Aurror ora, a, CO; CO; 4De Bioengineering, University Department D part pa r ment of Molec Molecular c Biology and Aarhus University, Aarhus, d Genetics, Aarhu huss Un hu U iver iv ersi er s ty si y, Aa Aarh rhus rh uss, Denmark; D nm De nmar arkk; 6In ar Institut nst stit ittut de de Biologie B ol Bi olog ogie og ie du du Developpement Developpem m de Marseille Aix-Marseille University, M arsei eill ei llee Luminy ll Lu umi m ny y ((IBDML), IB BDM DML) L , Ai L) A x-Ma M rs Ma rsei e ll ei llee Un niv iver ersi siity ty, Marseille, M rs Ma rsei e ll ei lle, e,, France Fraanc n e *contributed equally * on *c ontr trib ib but uted ed d eq equa quallly y
Correspondence: Russell Bowler, MD, PhD National Jewish Health 1400 Jackson Street, Room K715a Denver, CO 80206 Tel: 303-398-1639 Fax: 303-270-2249 E-mail: [email protected]
Journal Subject Codes: [156] Pulmonary biology and circulation, [91] Oxidant stress, [18] Pulmonary circulation and disease, [130] Animal models of human disease, [89] Genetics of cardiovascular disease 1 Downloaded from http://circgenetics.ahajournals.org/ at Queen's University--Kingston (CANADA) on August 29, 2014
DOI: 10.1161/CIRCGENETICS.113.000504
Abstract: Background - The enzyme extracellular superoxide dismutase (EC-SOD; SOD3) is a major antioxidant defense in lung and vasculature. A nonsynonomous single nucleotide polymorphism (SNP) in EC-SOD (rs1799895) leads to an arginine to glycine (Arg->Gly) amino acid substitution at position 213 (R213G) in the heparin-binding domain (HBD). In recent human genetic association studies, this SNP attenuates the risk of lung disease, yet paradoxically increases the risk of cardiovascular disease. Methods and Results - Capitalizing on the complete sequence homology between human and mouse in the HBD, we created an analogous R213G SNP knockin mouse. The R213G SNP did not change enzyme activity, but shifted the distribution of EC-SOD from om lung lung and and vvascular ascu as ular tissue to extracellular fluid (e.g. bronchoalveolar lavage fluid (BALF) and plasma). plas pl asma as ma). ma ). This Thi hiss sh sshift reduces susceptibility increases sceptibility scep pti t biiliity to to lung lu ung disease (lipopolysaccharide-induced (lipopolysacccha h ri r de-induced lung lunng injury) and increase susceptibility cardiopulmonary hypertension). i y tto ity o cardiop pul u mo mona nary ary ddisease i eaase is s ((chronic ch hroni n c hhypoxic ni yppoxi xic pu xi ppulmonary ulm monar a y hy ar yperten ten ensi s on si on). ) ). Conclusions optimal protection when localized n - We ns W conclude conncl c udde that th hat EC-SOD EC-S - OD pprovides roviides op optima m l pr prot tecctionn whe heen lo oca calize zeed to to the thh compartment oxidative n subjected to eextracellular nt xtraace xt cell llul ll ular a oxida d ti da t ve stress: str tres tr ess: thus, thuus, s thee redistribution red edis ed istr trib tr i ut utio on of EC-SOD D from the lung andd pulmonary p lmonaryy circulation pu circullatiion to the h extracellular extracell lllular l fluids fluid ds is beneficial benefficia i l iinn alveolar lungg disease but de discrepant detrimental detr trim imen im enta tall in pulmonary pul ulmo mona nary ry vascular vas ascu cula l r di la ddisease. seas se ase. e. T These hese he se ffindings i di in ding ngss account acco ac coun untt for f r the fo the discr disc di scrr risk associated with R213G in humans with lung diseases compared with cardiovascular diseases.
Key Words: pulmonary hypertension, lung, antioxidant enzymes, cardiovascular disease
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DOI: 10.1161/CIRCGENETICS.113.000504
Introduction In mammals, there are three superoxide dismutase (SOD) isoforms which constitute the major enzymatic antioxidant defense against superoxide. The extracellular isoform, EC-SOD or SOD3, is highly expressed in airways and lung epithelium and is the most abundant SOD in blood vessels. By reducing superoxide and oxidative stress, EC-SOD modulates multiple signaling pathways such as nitric oxide, NF-kappaB, Egr-1, and RhoA-dependent signal transduction 10-12. In animal studies using knockout and overexpressing mice, EC-SOD attenuates oxidative stress 13 19 and inflammation in the lungs from multiple inhaled toxins including LPS 4, 13-19 . Simila Similarly, arl rlyy in
the vasculature, EC-SOD preserves nitric oxide signaling, attenuates oxidative oxidattive in iinjury, j ry ju y, and an nd 20 3 21, 21 22 protects against a st vvascular ainst ascu as cula cu l r re remodeling 20,3, .
Because caausse animal studies stu tu udiees suggested sugggested e a role ed role for for EC EC-S EC-SOD SOD iinn cardiopulmonary cardio ca opulm lm monar aryy disease, ar disseasse, sseveral groups havee inve investigated vest ve stig st i ated ig d EC-SOD ECC SO SOD O gene gene assoc associations ocia oc iati ia t onns in ti in patients pat ati tient nts nt t with wiith heart hea eart aand nd lun lung ng di ddiseases. disease ise seas ase To date, 5 single g nucleotide po gle ppolymorphisms l morpphi ly hisms (S (SNPs) SNP NPs) s)) in in EC-SOD EC C-S SOD D (2 (2 coding codi dingg andd 3 non-coding) di non-codingg) have h 23-27 3 been associated cardiovascular disease processes 23 iatedd with iith th h clinical linii l ooutcomes tc tcomes iin cardio rdi dio asc llar or ppulmonary llmonar m di .
The nonsynonymous SNP (rs1799895) holds the most promise as a polymorphism that directly correlates with disease severity in humans and can provide insight into the mechanism for protection by EC-SOD. The rs1799895 SNP is a cytosine to guanine change at base pair +760 that results in an arginine to glycine substitution at amino acid 213 (R213G). The mean allele frequency of the R213G SNP is 4-6% in Asian populations and 2-3% in European populations 28, 29
. The R213G SNP was first reported to confer increased risk of ischemic heart disease in 9,188
participants from The Copenhagen City Heart Study29. Paradoxically, the R213G SNP was later found to reduce the risk of COPD and acute exacerbations of COPD in three independent, large population studies 23-25. Because the R213G SNP only occurs in humans, we have limited
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DOI: 10.1161/CIRCGENETICS.113.000504
experimental evidence and knowledge to explain this paradox of increased risk of vascular disease and reduced risk of pulmonary disease. From observational studies, we know that carriers of the R213G SNP have increased EC-SOD protein in plasma 30 and that the R213G variant EC-SOD protein has impaired in vitro binding to extracellular matrix elements 1; however, we do not know how the R213G SNP changes the distribution of EC-SOD in tissue nor do we understand how the R213G SNP alters the response to lung and vascular injury in vivo.
Methods Experimental animals: R213G SNP knockin mouse creation G knockin knoccki kinn targeting taarg get eting vector was created using n high fidelity Red/ET Red e /ET recombineering recombineerin The R213G riieffly, a 10.55 kkb b re regi gion gi onn w as sub ubbclloned e fro ed om a po osi s ti tivvely ly iidentified dentif de iffie iedd C5 C57B 7B BL/ L 6 BA B A methods. Briefly, region was subcloned from positively C57BL/6 BAC g homologous hoomo molo logo lo go ous recombination. rec ecom om mbi b naati tion on.. The on Th he short shor sh ortt hhomology or omo molo ogy arm arm r (SA) (SA S ) extended e te ex tend n ed 22.25 nd .225 kb k 33’ to clone using lanked Neo cas cassette sse sett ttee an tt and nd lo long ng hhomology omol om olog ol ogy arm ogy arm (LA) (LA) eextended xten xt en nde dedd 6. 6.13 133 kkb b to the 3’-end of the LoxP-flanked Lox ox P site. sit ite. e. The The he ssingle ingl in gle le Lo L Lox x P site site is is inserted inse in sert rtted upstream ups pstr t ea tr eam m of eexon xon 2 in in intron intr in tron on 1-2, 1-22, and and the the single L LoxP-flanked Neo cassette is inserted downstream of exon 2 in intron 2-3. The C -> G mutation (amino acid change: R>G) within exon 2 was generated by 3-step PCR mutagenesis. Using conventional sub cloning methods, the wild type sequence was replaced with the PCR fragment carrying the point mutation. Targeted iTL BA1 (C57BL/6N x 129/SvEv) hybrid embryonic stem cells were microinjected into C57BL/6 blastocysts. Resulting chimeras with a high percentage agouti coat color were mated to wild-type C57BL/6N mice to generate F1 heterozygous offspring. We obtained two independent C57BL/6 founder lines to generate homozygote R213G mice. DNA was sequenced periodically to verify lack of new spontaneous mutations. Animal experiments were approved by the Institutional Animal Care and Use Committee.
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Southern blot analysis DNA digested with BspEI was electrophoretically separated on a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with a probe targeted against the 5’ external region of ECSOD. DNA from C57BL/6, 129/SvEv, and BA1 (C57BL/6 x 129/SvEv) mice were used as wildtype controls. Mouse DNA genotyping R213G mice were genotyped using primers (Forward: 5’AGGCTCAAGTCTGTGCCAGAAGG-3’, Reverse: 5’TTTCCAATTCATTCACACACATGGG-3’) to distinguish wild type (357 7 bbpp amplicon) amplliconn) ffrom r knockin alleles (419 amplified eles ele es (4 4199 bbp p am amplicon). DNA was ampli lifi li f ed fi e in genoty genotyping ypi p ngg P PCR CR cocktail (10 mM primers, HiFi Buffer, mM MgSO4, minute iFi B iF uffer, 50 m M Mg M gSO O4, 10 10 mM mM DNTPs, DNTPs Ps, HiFi HiFi Taq, Taq q, water) watterr) by by a 5 m inu nute 99 99 oC hot less, 330 le 0 se econdd 995 5 oC denaturing, den de naturiing ng, 30 3 second sec econ ondd 55 55 oC annealing, an nneal alin lin i g, aand ndd 3 m minute inutte 68 inut 68 oC start for 30 cycle cycles, second extending conditions. c PCR R pr pproducts odducts were run on a 11.5% .5% 5% ag agarose garose ggel ell wi with ith a D DNA NA ladder. Human DNA NA N A genotyping t i Lungs, graciously provided by Dr. Mason (National Jewish Health), were donated through the National Disease Research Interchange (NDRI, Philadelphia, PA) and International Institute for the Advancement of Medicine (IIAM, Edison, NJ) under a Human Subject Research exempt protocol. Isolated lung DNA (Qiagen DNA tissue kit) was screened for the R213G SNP using Real-time PCR allelic discrimination assay-by-design service and the TaqMan SNP Genotyping Assay for Human SOD3 (C_2307506_10)(Applied Biosystems Life Technologies, Foster City, CA) DNA sequencing A DNA region containing the R213G SNP was sequenced using a forward primer (5’-
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AGGCTCAAGTCTG TGCCAGAAGG-3’) located in the long homology arm upstream of the R213G SNP and a reverse primer (5’-GGAACTTCGCTAGA CTAGTACGCGTG-3’) located within the Neo cassette downstream of the R213G SNP per established protocols (Molecular Research Center, National Jewish Health). DNA was amplified and the PCR product sequenced on an ABI PRISM 3100 genetic analyzer. pGEM-3zf(+) DNA was used as a positive control. EC-SOD activity assays Pulverized lung (70 mg) was homogenized in SOD activity assay buffer and total lung protein concentration was determined by BCA protein assay (Pierce Biotechnology, ology, y Rockford, IL). IL) L ___ EC-SOD was separated from intracellular SOD (SOD1 and SOD2) using ing the th he Glycoprotein Glycopprote Gl tein te in Isolation Kit, following it, ConA Con onA A (Pierce (P Pierc rcee Biotechnology) per manufacturer’s rc man anuf an u acturer’s instru instructions uct ctions with the follow minor modifications: 300 Lectin Resin the intracellular ifications: spinn columns if colu umnss contained conttainedd 30 00 ȝll ConA ConA nA L ectiin Re esiin aand nd th he in ntrraceelllul SOD fraction washing with o was on ass collected col ollect ctted d pprior rior ri ior to wa ashingg colu ccolumns. olu l mn mnss. Columns Collum umnns were wer eree inc iincubated ncuba bate ba tedd wi te ith h 3300 00 ȝl 00 ȝ elution buffer f for 15 minutes prior fer p ior to collection pr coll llectio i n off flow-through. flo l w-througgh. h Absence A sence of EC-SOD in the Ab t intracellular SOD blott while containing SOD ffraction tii was confirmed fi d by b western t bl hil hil eluted l t d fractions f tii t i i ECSOD were pooled and concentrated 10-fold using a centriprep concentrator (Amicon-ultracel10K, Millipore). Activity levels were measured by the SOD assay kit-WST (Dojindo Molecular Technologies, Maryland, USA), according to manufacturer’s instructions. Data were expressed as units of EC-SOD activity per mg of total lung protein. Heparin-sepharose binding Pooled plasma or lung tissue from ten mice (wild type or R213G) was homogenized and centrifuged; and supernatant dialyzed against 20 mM Tris –HCl pH 7.5 1. Following dialysis, the sample was applied to 1ml HiTrap Heparin Sepharose (GE Healthcare) and heparin-binding proteins eluted with a linear gradient of NaCl (0 mM±1 M) in 50 mM Tris-HCl, pH 7.5 at a flow
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rate of 1ml/min. Fractions were analyzed for EC-SOD activity by inhibition of cytochrome c reduction as described previously 2. To test if the R213G SNP altered SOD activity, we normalized activity to the relative EC-SOD content in the peak fraction. We determined the relative EC-SOD content by western blotting using a polyclonal rabbit murine EC-SOD antibody and Cy5 labeled anti-rabbit secondary antibody (Sigma). Histological analysis EC-SOD immunostaining was performed as previously described using mouse- or humanspecific antibodies. 3,4, 5. Mouse lungs were inflated-fixed at 25 cm H20 pressure with 4% % paraformaldehyde. EC-SOD KO and wild type mice were used as negative ative aand ndd posit positive itiivee ccontrols it on respectively. y y. Bronchoalveolar veo olar lavagee Lungs weree lavag lavaged ag ged wit with th 1 mL LP PBS BS S and and 200 μll of of total to otal tal bronchoalveolar b on br onch nch chooalv lveo lv eola eola l r la llavage vaage g ffluid lu uid (BALF) ((BALF BA BAL ALF was diluted in 100 mL Isoton II diluent dil iluent and andd countedd on a Z Z2 2 pa pparticle rticlle count and andd size siize analyzer analyz y er (Beckman C Co Coulter, lter lt F Fullerton, llerton ll to CA) CA). CA) Western blot analysis Homogenized lung tissue was run on a 12% Tris/Glycine SDS-PAGE gel and transferred to PVDF membrane per standard protocols 6. Blots were incubated with primary antibody as follows: Beta-actin 1:20,000 (Sigma, St. Louis, MO), EC-SOD 1:1000 (8130), 4-HNE 1:1000 (Abcam, Cambridge, MA), nitrotyrosine 1:500 (UpState, Lake Placid, NY). Following incubation with secondary antibody, blots were developed with ECL Plus (GE), imaged with STORM 860 (GE) using the fluorescent blue (430nm) setting, quantified using ImageQuant software, and expressed relative to beta-actin.
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Gene expression Total RNA was extracted using either TRIzol (Invitrogen, Carslbad CA) or RNeasy kit (Qiagen) according to the manufacturer’s instructions and differences in gene expression were determined using Real-Time RT-PCR as previously described 7. Gene expression was determined using FRPSDUDWLYHWKUHVKROGF\FOH ǻǻ&T) as suggested by the manufacturer (Applied Biosystems, Foster City, CA) normalizing each sample to 18s rRNA. The following primer sets were purchased from Assays on Demand (Applied Biosystems); 18s rRNA (cat#4310893E), SOD3 (Mm01213380-s1), SOD1 (Mm01700393_g1) and SOD2 (Mm00449726_m1). 726_m1). Proinflammatory cytokine and chemokine measurements n cchemokine nd h mok he okin i e cconcentrations oncentrations in mouse lu lun ng tissue and BALF BA ALF L were measured (ELISA (E E Cytokine and lung roraa, CO) usin ro ng eith th her the h MSD he S m SD ouusee pr roinfl flam fl am mma m to ory 7-plex x ultra a-ssens nsitiv ve kkit or TECH, Aurora, using either mouse proinflammatory 7-plex ultra-sensitive a ammat ator at oryy panel or pane nell 1 V-PLEX V PL VP EX kkit it (M Mes esoo Scale Scalle Discovery, Scal Disc Di scov sc overy, ov y Gaithersburg, Gaiith the hersb sbur sb u g, MD) ur MD) D) per per the proinflammatory (Meso r rer’s instructions. manufacturer’s tii l cell llll counts t Total differential Total BALF was centrifuged for 15 minutes at 300 X g. The cell pellet was resuspended in 200 ul PBS. 180 ul was spotted to glass slides and allowed to dry overnight. Slides were then stained for differential cell counts using HEMA 3 stain per manufacturer’s instruction (Fisher Scientific, Kalamazoo, MI). Chronic hypoxic pulmonary hypertension Four week old WT C57/BL6 and HM R213G mice were exposed to hypobaric hypoxia (395 torr) for 35 days: chambers simulated altitude (5486 m) equivalent to 10% oxygen. Normoxic control mice were maintained in Denver ambient air (1,609 m).
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Isolation of pulmonary arteries Lungs were perfused with a slurry of iron particles in warmed agarose and minced; ironcontaining PAs were separated from lung parenchyma by magnet.8 The PA was digested with collagenase to release the iron particles, and the isolated PA tissue homogenized and sonicated in RIPA buffer for western blots. Assessment of pulmonary hypertension Right ventricular systolic pressure (RVSP) was measured in anesthetized mice by direct RV puncture through a closed chest using the Cardiomax III Cardiac Output ut system (C ((Columbus olum mbu bus Instruments) as previously described 7. Right ventricular hypertrophy (RVH) (RVH H) was de ddetermined term min as ght h vventricular/left entr en tric tr i ul ulaar/left ventricular + septum um weights (RV/LV+ V+S). V+ the ratio of rig right (RV/LV+S). Assessment nt off pulmonar pulmonary ry vascular vaascullar a remodeling rem emod odelin ng poxi po xiaa-indu xi d ce du cedd mu muscul ular ul ari ar rizatio on of sma mall lll ves essse sels ls, lu ls ung ng sections sec ecti t onns were weree sstained tain tain i ed dw To evaluatee hypo hypoxia-induced muscularization small vessels, lung with PRXVHPRQRFORQDOĮ-VPRRWKPXVFOHDFWLQDQWLERG\ Į-SMA Q QRFORQDO Į-VPRRWK KPXVFOHDFWL O LQDQWLER E GG\\ Į-S SMA A )(1:100, )(1 (1:1 100 00,, Clone Cl 1A4)) and the number of positi positively siitii ell stained taiined d small ll vessels essels ls ((gly extracellular superoxide dismutase (ec-sod) in plasma is caused by a reduction of both heparin and collagen affinities. Biochem J. 2005;385:427-432. 2. Oury TD, Crapo JD, Valnickova Z, Enghild JJ. Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: A simplified, high-yield purification of extracellular superoxide dismutase. Biochem J. 1996;317( Pt 1):51-57. 3. Nozik-Grayck E, Suliman HB, Majka S, Albietz J, Van Rheen Z, Roush K, et al. Lung ec-sod overexpression attenuates hypoxic induction of egr-1 and chronic hypoxic pulmonary vascular remodeling. Am J Physiol Lung Cell Mol Physiol. 2008;295:L422-430.
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4. Bowler RP, Nicks M, Warnick K, Crapo JD. Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2002;282:L719726. 5. Oury TD, Schaefer LM, Fattman CL, Choi A, Weck KE, Watkins SC. Depletion of pulmonary ec-sod after exposure to hyperoxia. Am J Physiol Lung Cell Mol Physiol. 2002;283:L777-784. 6. Villegas LR, Kluck D, Field C, Oberley-Deegan RE, Woods C, Yeager ME, et al. Superoxide dismutase mimetic, mnte-2-pyp, attenuates chronic hypoxia-induced pulmonary hypertension, pulmonary vascular remodeling, and activation of the nalp3 inflammasome. Antioxid Redox Signal. 2013;18:1753-1764 7. Hartney JM, Brown J, Chu HW, Chang LY, Pelanda R, Torres RM. Arhgef1 regulates alpha5beta1 integrin-mediated matrix metalloproteinase expression and is required for homeostatic lung immunity. Am J Pathol. 2010;176:1157-1168. 8. Waypa GB, Chandel NS, Schumacker PT. Model for hypoxic pulmonary vasoconstriction onaryy vasoconstric icti ic tio ti involving mitochondrial oxygen sensing. Circ Res. 2001;88:1259-1266. 6 9. Lim RS, Kratzer Barry NP, Miyazaki-Anzai Kraatzer A, Bar arrry N arry P, M P, iyaz iy azak az ak ki-A Anz nzai a S,, Miyazaki Miya yaaza yaza zaki k M, M, Mantulin Mant Ma n ulin n WW, WW, W, et al. al. Multimodall cars caars microscopy microsco opy determination determi m nati t onn of ti of the th he impact im mpact ct of of diet diett on on macrophage maacrop opphagge ge infiltration innfiil il atiion and iltra lipid accumulation formation mice. Lipid 2010;51:1729-1737. mula mu lati tion on pl pplaque aq que fo orma mati ma tion iin n ap aapoe-deficient poee-ddefiiciien nt mi mice e. J L ipi p d Res. pi Rees. 2010 10;5 10 51:17729-10. Richenss TR, Linderman Horstmann SA, Lambert Xiao Keith Cigarette Lindermaan DJ DJ, Ho H rstm rs tman tm an nn SA A, La Lamb mb ber e t C, X iaoo YQ ia YQ,, Ke Keit ithh RL, et al. Ciga it a smoke impairs apoptotic cells through activation Am J a clearance off apo airs p pt p otic i cel lls th hrough ghh oxidant-dependent oxiidant-ddep pende d nt acti ivation of rhoa. A Respir Crit Care Med. Med ed. d. 20 22009;179:1011-1021. 09;1 09 ;1 179:1 79:1 79 1011011-10 01 1-10 1021 21. 21 11. Bowler RP, Barnes PJ, Crapo JD. The role of oxidative stress in chronic obstructive pulmonary disease. Copd. 2004;1:255-277. 12. Jung O, Marklund SL, Geiger H, Pedrazzini T, Busse R, Brandes RP. Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: In vivo and ex vivo evidence from ecsod-deficient mice. Circ Res. 2003;93:622-629. 13. Bowler RP, Arcaroli J, Crapo JD, Ross A, Slot JW, Abraham E. Extracellular superoxide dismutase attenuates lung injury after hemorrhage. Am J Respir Crit Care Med. 2001;164:290294. 14. Bowler RP, Arcaroli J, Abraham E, Patel M, Chang LY, Crapo JD. Evidence for extracellular superoxide dismutase as a mediator of hemorrhage-induced lung injury. Am J Physiol Lung Cell Mol Physiol. 2003;284:L680-687. 15. Bowler RP, Nicks M, Tran K, Tanner G, Chang LY, Young SK, et al. Extracellular superoxide dismutase attenuates lipopolysaccharide-induced neutrophilic inflammation. Am J Respir Cell Mol Biol. 2004;31:432-439.
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16. Tollefson AK, Oberley-Deegan RE, Butterfield KT, Nicks ME, Weaver MR, Remigio LK, et al. Endogenous enzymes (nox and ecsod) regulate smoke-induced oxidative stress. Free Radic Biol Med. 2010;49:1937-1946. 17. Yao H, Arunachalam G, Hwang JW, Chung S, Sundar IK, Kinnula VL, et al. Extracellular superoxide dismutase protects against pulmonary emphysema by attenuating oxidative fragmentation of ecm. Proc Natl Acad Sci U S A. 2010. 18. Ghio AJ, Suliman HB, Carter JD, Abushamaa AM, Folz RJ. Overexpression of extracellular superoxide dismutase decreases lung injury after exposure to oil fly ash. Am J Physiol Lung Cell Mol Physiol. 2002;283:L211-218. 19. Kang SK, Rabbani ZN, Folz RJ, Golson ML, Huang H, Yu D, et al. Overexpression of extracellular superoxide dismutase protects mice from radiation-induced lung injury. Int J Radiat Oncol Biol Phys. 2003;57:1056-1066. 20. Oury TD, Ho YS, Piantadosi CA, Crapo JD. Extracellular superoxide dismutase, nitric ide di dismut tase,, nit itri it ric oxide, ri and central nervous system o2 toxicity. Proc Natl Acad Sci U S A. 1992;89:9715-9719. 92;89::97 9715 15 971 7199 21. Hartney Venkataraman Villegas y T, Birari R, R, V e ka en kata tara ta raama mann S, S V i leega il gas L, L, Martinez Mar arrti arti tine n z M, ne M Black Black laac SM, SM, M, et et al. all Xanthine Xan anthi oxidase-derived upregulate egr-1 erk1/2 smooth muscle cells; model test r veed ros upregu rive gu ulatte egr r-1 viaa er rk1/2 inn paa smo moot othh mu ot uscle ce ellss; mode del ttoo tes de st impact im m of extracellular ullar ros in ch cchronic ronnic hhypoxia. ypooxi xia. PLoS PLoS o ONE. ON NE E. 2011;6:e27531. 20111;6 20 ;6:ee275531. 22. Xu Y, Stenmark KR, D smooth Das ass M M,, Wa W Walchak lccha h k SJ S SJ,, Ru R Ruff ff L LJ, J, D Dempsey em mpsey pssey E EC. C P C. Pulmonary ulmo ul monnary artery sm mo muscle cellss from chronicall chronically hypoxic l y hhy ll ypo p xic neonatal neonatall calves callves retain ffetal-like etal-l l like ik k andd acquire acqu q ire new gr ggrowth o properties. A Am Physiol. 1997;273:L234-245. m J Phy ysi siol ol.. 19 ol 1997 977;273 97;2 273 73:L :L23 :L23 2344 24 245. 5. 23. Juul K, Tybjaerg-Hansen A, Marklund S, Lange P, Nordestgaard BG. Genetically increased antioxidative protection and decreased chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2006;173:858-864. 24. Young RP, Hopkins R, Black PN, Eddy C, Wu L, Gamble GD, et al. Functional variants of antioxidant genes in smokers with copd and in those with normal lung function. Thorax. 2006;61:394-399. 25. Siedlinski M, van Diemen CC, Postma DS, Vonk JM, Boezen HM. Superoxide dismutases, lung function and bronchial responsiveness in a general population. Eur Respir J. 2009;33:986992. 26. Dahl M, Bowler RP, Juul K, Crapo JD, Levy S, Nordestgaard BG. Superoxide dismutase 3 polymorphism associated with reduced lung function in two large populations. Am J Respir Crit Care Med. 2008;178:906-912. 27. Sorheim IC, DeMeo DL, Washko G, Litonjua A, Sparrow D, Bowler R, et al. Polymorphisms in the superoxide dismutase-3 gene are associated with emphysema in copd. COPD. 2010;7:262-268.
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28. Marklund SL, Nilsson P, Israelsson K, Schampi I, Peltonen M, Asplund K. Two variants of extracellular-superoxide dismutase: Relationship to cardiovascular risk factors in an unselected middle-aged population. J Intern Med. 1997;242:5-14. 29. Juul K, Tybjaerg-Hansen A, Marklund S, Heegaard NH, Steffensen R, Sillesen H, et al. Genetically reduced antioxidative protection and increased ischemic heart disease risk: The copenhagen city heart study. Circulation. 2004;109:59-65. 30. Sandstrom J, Nilsson P, Karlsson K, Marklund SL. 10-fold increase in human plasma extracellular superoxide dismutase content caused by a mutation in heparin-binding domain. J Biol Chem. 1994;269:19163-19166
Figure Legends:
Figure 1. Characterization R213G SNP knockin mouse. recombination Char Ch arac a terizaati t onn off R 2133G SN NP kn nocckin mo m use. ((A) A) Homologous Homo Ho olo ogo gouus rec ecom ec ombbinaatiionn om introduces the SNP identical t identical R213G R21 2 3G G (CoG) (CoG) S NP rresulting esul es ulti ul ting ti ng iin n an n ide deenntticcal aarginine r in rg nin inee to glycine amino am m acids substitution itution in the it th he extracellular ex xtr trac accel e lu ula lar matrix-binding matr ma trix ix x-b -bin bin indi ding di ng domain dom omai aiin (ECM-BD) ain (ECM (E ECM M-BD BD)) off EC-SOD. EC-SO SOD. Bluee amino acids represent the conserved ECM-BD region and red represent R213G SNP at position 213. (B) R213G SNP homologous recombinant containing a Neo cassette (Neo). Red asterisk is site of R213G SNP. Black triangles are LoxP sites. PB ½ is probe site for Southern blots. (C) Confirmation of R213G positive clones (asterisks) by Southern blot. (D) R213G mouse DNA genotyping. Wild type (WT) mice produce a 357 bp amplicon, R213G heterozygous (HET) mice produce both a 357 bp and 419 bp amplicon, and R213G homozygous (HM) mice produce 419 bp amplicon. (E) DNA sequence confirms CoG mutation (red asterisk) changing the codon from CGG (Arginine) to GGG (Glycine). (F) Plasma EC-SOD protein of WT, HET and HM mice. (G) EC-SOD activity in units per ml of plasma (n = 6 for all genotypes). (H) Heparin sepharose binding using purified EC-SOD from wild type (WT, n = 1) open squares and dashed
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line) and R213G homozygous (R213G, n = 1) mouse (black squares and solid line) in units/ml (U/ml) of elution volume. (I) SOD activity normalized to protein (n =3 for both WT and R213G). (J) EC-SOD immunostaining in murine lung tissue. Brown staining (arrow = airway, arrowhead = vessel) for wild type (WT), R213G heterozygous (HET) and R213G homozygous (HM) mice. EC-SOD knockout mice (KO) were used as background EC-SOD staining controls. (K) EC-SOD immunostaining in mouse aorta. Brown staining (arrows) shows EC-SOD localization for WT, HET and HM mice. EC-SOD immunodepleted (ID) and Phosphate buffered saline (PBS) were used for nonspecific background staining detection. ((L) total L) EC-SOD tota al pr pprotein was analyzed in total lung tissue by western blot from WT (n = 3), HET HM ET (n = 3) aand ndd H M (n (n =3) animals. (M) EC-SOD M EC M) EC-S -SOD SOD activity act cttiv i ity in units per mg of lung lun ungg tissue protein of un of WT (n = 6), Het (n = 6) and HM (n = 55)) mice. (N) EC EC-SOD protein lung WT and R213G EC-S SOD D prote t in te n iin n lu ung tissue tisssuee fr from o W om T an nd R2 213G G human huma m n carrier. caarrr (O) EC-SOD wild R213G OD protein pro rote ro tein te i in bronchoalveolar in b on br onch ch hoa oalveo eo ola lar lavage lava vagge fluid va fluid luid (BALF) (BA B LF LF)) off w ildd type il typpe (WT) ty (WT WT) an andd R2 R R213 213 13 homozygous EC-SOD R213G u (R213G) us (R213G)) mice. (P) (P) EC E -S SOD O protein proteiin in in BALF BA ALF from from WT aand ndd R 213G human carrier caa obtained post-mortem. Data as mean r S S.E. WT. t t D t are shown h E * P < 0.05 0 05 compared d tto WT
Figure 2. Mice with R213G SNP have attenuated injury to inhaled LPS. (A) Total cells counts in BALF 0, 4 and 24 hours post-LPS. (n = 4 per group). (B) Total neutrophil counts in BALF (C) Total macrophages counts in BALF. (D) Cytokines in BALF of WT and R213G animals at 0 hours (WT n = 4, R213G n = 3), 4 hours (WT n = 8, R213G n= 4) and 24 hours (WT n = 9, R213G n = 4) post LPS inhalation. (E) 4-HNE in lung tissue. Data are presented as mean r S.E. # P < 0.05 compared to samples at 0 hours for each genotype. * P < 0.05 comparing WT to R213G samples under identical conditions.
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Figure 3. R213G SNP reduces vascular EC-SOD and increases susceptibility to pulmonary hypertension. (A) EC-SOD protein expression in pulmonary artery tissue isolated from normoxic (NX) wild type (WT) and R213G homozygous mice (R213G). (B) Right ventricular systolic pressure (RVSP mmHg) at baseline (WT n = 9, R213G n = 6) and after 35 days of chronic hypoxia (HPX) in WT (n = 8) and R213G (n = 6). (C) Right ventricular hypertrophy (RV/LV+S weights) at baseline (WT n = 8, R213G n = 6) after 35 days of HPX (WT n = 7, R213G n = 8). (D) Representative lung Į-sma immunostaining (brown signal) with counterstain by hematoxylin (blue) after 35 days of HPX. Bar = 50 microns. Arrows indicate muscularized vessels ularized small ves sse sels LGHQWLILHGE\SRVLWLYHĮ-sma immunostaining. (E) Quantitation of muscularized culariizedd small smalll vessels veesse (< 50 microns)) inn a 10x R213G after 10x 0 field fie i ld d at at baseline (WT n = 5, R2 R213 1 G n = 6) and af aft ter 35 days of HPX ((WT n = 4, R213G Collagen G n = 5). (F) Col llag gen visualized vis isualiizedd by is by TPE-SHG TPE--SHG G inn representative reeprresenttattive ve unstained unsta t in ta i ed d tissue tisssuee sections. Collagen signal), autofluorescence signal). o en ollagen n (red (red si ign gnal all), ttissue i suee au is autofl luo uore reescen ence ce ((green grree eenn sign gn nal al). l) (G) (G) Quantification Qu ifi Qu Quanti fica cati tiion of tion o collagen standardized a andardized for vessel size (n= ( = 6 for all groups). (n g oups gr p ). ) Data are shown as mean r S.E. * P < 0.01 for WT identical T compared d to R213G samples l under d id i l conditions. di i # P < 00.01 01 for f NX compared to HPX.
Figure 4. Schematic demonstrating the change in distribution of EC-SOD associated with the R213 SNP. The consequence of the change in distribution is a reduction in susceptibility to inflammation from lipopolysaccharide (LPS) and increased susceptibility to vascular disease (hypoxic pulmonary hypertension and remodeling).
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SUPPLEMENTAL MATERIAL Supplemental Methods Gene expression Total RNA was extracted using either TRIzol (Invitrogen, Carlbad, CA or RNeasy kit (Qiagen) according to the manufacturer’s instructions and differences in gene expression were determined using Real-Time RT-PCR as previously described1. Gene expression was determined using comparative threshold cycle (ΔΔCT) as suggested by the manufacturer (Applied Biosystems, Foster City, CA) normalizing each sample to 18s rRNA. The following primer sets were purchased from Assays on Demand (Applied Biosystems): 18s rRNA (cat #4310893E) and SOD3 (cat# Mm01213380-s1). Proinflammatory cytokine and chemokine measurements Cytokine and chemokine concentration in mouse lung tissue were measured using the MSD (MesoScale Discovery, Gaithersburg, MD) Proinflammatory Panel 1 V-PLEX kit. Total protein in bronchoalveolar lavage Total protein was quantitated using Coomassie Plus (Bradford) Assay kit (Thermo Fisher Scientific, Rockford, IL) per manufacturer’s instructions. Supplemental Figures
Figure legends Fig 1. (A) Expression of SOD3 was measured by Real-Time RT-PCR in whole lung tissue from WT (n = 3), HET (n = 3) and HM (n = 3) animals. Gene expression was normalized to 18s rRNA
expression and displayed relative to WT samples. (B) Expression of SOD3 was measured by Real-Time TR-PCR in isolated aortas from WT (n = 3), HET (n = 3) and HM (n = 3) animals. Gene expression was normalized to 18s rRNA expression and displayed relative to WT samples. (C) Proinflammatory cytokines and chemokines in lung tissue from WT and R213G animals at 0 (WT n = 4, R213G n = 3), 4 (WT n = 8, R213G n = 4) and 24 hours (WT n =9, R213G n = 4) post LPS inhalation. Mediators are expressed as pg per 30ug of total protein. (D) Total protein in broncoalveolar lavage fluid (BALF) in WT and R213G animals at 0 (WT n = 3, R213G n = 2), 4 (WT n = 7, R213G n = 4) and 24 hours (WT n = 4, R213G n = 7) post LPS inhalation. Data are presented as mean ± S.E. # P < 0.05 compared to samples at 0 hours for each genotype. * P < 0.05 comparing WT to R213G samples under identical conditions. Supplemental References 1.
Hartney JM, Brown J, Chu HW, Chang LY, Pelanda R, Torres RM. Arhgef1 regulates alpha5beta1 integrin-mediated matrix metalloproteinase expression and is required for homeostatic lung immunity. The American journal of pathology. 2010;176:1157-1168
Supplemental Figure 1 A
B
EC-SOD in Lung Relative Gene Expression
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DOI: 10.1161/CIRCGENETICS.113.000504
A Common Polymorphism in EC-SOD Affects Cardiopulmonary Disease Risk by Altering Protein Distribution
Running title: Hartney et al.; EC-SOD polymorphism alters distribution
John M. Hartney, PhD1,2*; Timothy Stidham, MD3*; David A. Goldstrohm, PhD1*; Rebecca E. Oberley-Deegan, PhD1; Michael R. Weaver, BS1; Zuzana Valnickova-Hansen, BSc4; Carsten Scavenius, PhD4; Richard K.P. Benninger, PhD3,5; Katelyn n F. L Leahy, eahy ea hy, BS1; Ri Rich Richard ch Johnson, BS3; Fabienne Gally, PhD1; Beata Kosmider, PhD1; Angelaa K. Zi Zimmermann Zimmermann, n, P PhD6; n J. Eng Enghild nghi gh ld d, Ph PhD hD4; Eva Nozik-Grayck, Nozik Grayck MD3*; Russell P. P Bowler, Bowler B o ler, MD, ow MD PhD1* Jan Enghild,
1
Department n of nt of Medicine, M dicine Me ne, Na ne National ation on nal Jew Jewish ewish He H Health; altth; 2In al Integrated ntegr grated ed Department Dep epartm tment off Immunology, Imm mm munol olog ol ogy, og y,, Un University nivverr of 3 5 Colorado Denver National Jewish Health, Denver; d De do Denv nv ver aand nd N atio at iona io nall Jewi na wish wi shh H ealt lthh, D lt enve verr; De ve Department Dep part rtme rt mentt of ment of Pediatrics, Peedi d at atri rics ri cs, De cs Department Depa part pa rtme rt ment me nt of e eering, Universi ity of of Colorado Colo Co lo ora rado do School Sch hoo o l of Medicine, Med edic iccin ine, e Aurora, Aurror ora, a, CO; CO; 4De Bioengineering, University Department D part pa r ment of Molec Molecular c Biology and Aarhus University, Aarhus, d Genetics, Aarhu huss Un hu U iver iv ersi er s ty si y, Aa Aarh rhus rh uss, Denmark; D nm De nmar arkk; 6In ar Institut nst stit ittut de de Biologie B ol Bi olog ogie og ie du du Developpement Developpem m de Marseille Aix-Marseille University, M arsei eill ei llee Luminy ll Lu umi m ny y ((IBDML), IB BDM DML) L , Ai L) A x-Ma M rs Ma rsei e ll ei llee Un niv iver ersi siity ty, Marseille, M rs Ma rsei e ll ei lle, e,, France Fraanc n e *contributed equally * on *c ontr trib ib but uted ed d eq equa quallly y
Correspondence: Russell Bowler, MD, PhD National Jewish Health 1400 Jackson Street, Room K715a Denver, CO 80206 Tel: 303-398-1639 Fax: 303-270-2249 E-mail: [email protected]
Journal Subject Codes: [156] Pulmonary biology and circulation, [91] Oxidant stress, [18] Pulmonary circulation and disease, [130] Animal models of human disease, [89] Genetics of cardiovascular disease 1 Downloaded from http://circgenetics.ahajournals.org/ at Queen's University--Kingston (CANADA) on August 29, 2014
DOI: 10.1161/CIRCGENETICS.113.000504
Abstract: Background - The enzyme extracellular superoxide dismutase (EC-SOD; SOD3) is a major antioxidant defense in lung and vasculature. A nonsynonomous single nucleotide polymorphism (SNP) in EC-SOD (rs1799895) leads to an arginine to glycine (Arg->Gly) amino acid substitution at position 213 (R213G) in the heparin-binding domain (HBD). In recent human genetic association studies, this SNP attenuates the risk of lung disease, yet paradoxically increases the risk of cardiovascular disease. Methods and Results - Capitalizing on the complete sequence homology between human and mouse in the HBD, we created an analogous R213G SNP knockin mouse. The R213G SNP did not change enzyme activity, but shifted the distribution of EC-SOD from om lung lung and and vvascular ascu as ular tissue to extracellular fluid (e.g. bronchoalveolar lavage fluid (BALF) and plasma). plas pl asma as ma). ma ). This Thi hiss sh sshift reduces susceptibility increases sceptibility scep pti t biiliity to to lung lu ung disease (lipopolysaccharide-induced (lipopolysacccha h ri r de-induced lung lunng injury) and increase susceptibility cardiopulmonary hypertension). i y tto ity o cardiop pul u mo mona nary ary ddisease i eaase is s ((chronic ch hroni n c hhypoxic ni yppoxi xic pu xi ppulmonary ulm monar a y hy ar yperten ten ensi s on si on). ) ). Conclusions optimal protection when localized n - We ns W conclude conncl c udde that th hat EC-SOD EC-S - OD pprovides roviides op optima m l pr prot tecctionn whe heen lo oca calize zeed to to the thh compartment oxidative n subjected to eextracellular nt xtraace xt cell llul ll ular a oxida d ti da t ve stress: str tres tr ess: thus, thuus, s thee redistribution red edis ed istr trib tr i ut utio on of EC-SOD D from the lung andd pulmonary p lmonaryy circulation pu circullatiion to the h extracellular extracell lllular l fluids fluid ds is beneficial benefficia i l iinn alveolar lungg disease but de discrepant detrimental detr trim imen im enta tall in pulmonary pul ulmo mona nary ry vascular vas ascu cula l r di la ddisease. seas se ase. e. T These hese he se ffindings i di in ding ngss account acco ac coun untt for f r the fo the discr disc di scrr risk associated with R213G in humans with lung diseases compared with cardiovascular diseases.
Key Words: pulmonary hypertension, lung, antioxidant enzymes, cardiovascular disease
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Introduction In mammals, there are three superoxide dismutase (SOD) isoforms which constitute the major enzymatic antioxidant defense against superoxide. The extracellular isoform, EC-SOD or SOD3, is highly expressed in airways and lung epithelium and is the most abundant SOD in blood vessels. By reducing superoxide and oxidative stress, EC-SOD modulates multiple signaling pathways such as nitric oxide, NF-kappaB, Egr-1, and RhoA-dependent signal transduction 10-12. In animal studies using knockout and overexpressing mice, EC-SOD attenuates oxidative stress 13 19 and inflammation in the lungs from multiple inhaled toxins including LPS 4, 13-19 . Simila Similarly, arl rlyy in
the vasculature, EC-SOD preserves nitric oxide signaling, attenuates oxidative oxidattive in iinjury, j ry ju y, and an nd 20 3 21, 21 22 protects against a st vvascular ainst ascu as cula cu l r re remodeling 20,3, .
Because caausse animal studies stu tu udiees suggested sugggested e a role ed role for for EC EC-S EC-SOD SOD iinn cardiopulmonary cardio ca opulm lm monar aryy disease, ar disseasse, sseveral groups havee inve investigated vest ve stig st i ated ig d EC-SOD ECC SO SOD O gene gene assoc associations ocia oc iati ia t onns in ti in patients pat ati tient nts nt t with wiith heart hea eart aand nd lun lung ng di ddiseases. disease ise seas ase To date, 5 single g nucleotide po gle ppolymorphisms l morpphi ly hisms (S (SNPs) SNP NPs) s)) in in EC-SOD EC C-S SOD D (2 (2 coding codi dingg andd 3 non-coding) di non-codingg) have h 23-27 3 been associated cardiovascular disease processes 23 iatedd with iith th h clinical linii l ooutcomes tc tcomes iin cardio rdi dio asc llar or ppulmonary llmonar m di .
The nonsynonymous SNP (rs1799895) holds the most promise as a polymorphism that directly correlates with disease severity in humans and can provide insight into the mechanism for protection by EC-SOD. The rs1799895 SNP is a cytosine to guanine change at base pair +760 that results in an arginine to glycine substitution at amino acid 213 (R213G). The mean allele frequency of the R213G SNP is 4-6% in Asian populations and 2-3% in European populations 28, 29
. The R213G SNP was first reported to confer increased risk of ischemic heart disease in 9,188
participants from The Copenhagen City Heart Study29. Paradoxically, the R213G SNP was later found to reduce the risk of COPD and acute exacerbations of COPD in three independent, large population studies 23-25. Because the R213G SNP only occurs in humans, we have limited
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DOI: 10.1161/CIRCGENETICS.113.000504
experimental evidence and knowledge to explain this paradox of increased risk of vascular disease and reduced risk of pulmonary disease. From observational studies, we know that carriers of the R213G SNP have increased EC-SOD protein in plasma 30 and that the R213G variant EC-SOD protein has impaired in vitro binding to extracellular matrix elements 1; however, we do not know how the R213G SNP changes the distribution of EC-SOD in tissue nor do we understand how the R213G SNP alters the response to lung and vascular injury in vivo.
Methods Experimental animals: R213G SNP knockin mouse creation G knockin knoccki kinn targeting taarg get eting vector was created using n high fidelity Red/ET Red e /ET recombineering recombineerin The R213G riieffly, a 10.55 kkb b re regi gion gi onn w as sub ubbclloned e fro ed om a po osi s ti tivvely ly iidentified dentif de iffie iedd C5 C57B 7B BL/ L 6 BA B A methods. Briefly, region was subcloned from positively C57BL/6 BAC g homologous hoomo molo logo lo go ous recombination. rec ecom om mbi b naati tion on.. The on Th he short shor sh ortt hhomology or omo molo ogy arm arm r (SA) (SA S ) extended e te ex tend n ed 22.25 nd .225 kb k 33’ to clone using lanked Neo cas cassette sse sett ttee an tt and nd lo long ng hhomology omol om olog ol ogy arm ogy arm (LA) (LA) eextended xten xt en nde dedd 6. 6.13 133 kkb b to the 3’-end of the LoxP-flanked Lox ox P site. sit ite. e. The The he ssingle ingl in gle le Lo L Lox x P site site is is inserted inse in sert rtted upstream ups pstr t ea tr eam m of eexon xon 2 in in intron intr in tron on 1-2, 1-22, and and the the single L LoxP-flanked Neo cassette is inserted downstream of exon 2 in intron 2-3. The C -> G mutation (amino acid change: R>G) within exon 2 was generated by 3-step PCR mutagenesis. Using conventional sub cloning methods, the wild type sequence was replaced with the PCR fragment carrying the point mutation. Targeted iTL BA1 (C57BL/6N x 129/SvEv) hybrid embryonic stem cells were microinjected into C57BL/6 blastocysts. Resulting chimeras with a high percentage agouti coat color were mated to wild-type C57BL/6N mice to generate F1 heterozygous offspring. We obtained two independent C57BL/6 founder lines to generate homozygote R213G mice. DNA was sequenced periodically to verify lack of new spontaneous mutations. Animal experiments were approved by the Institutional Animal Care and Use Committee.
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Southern blot analysis DNA digested with BspEI was electrophoretically separated on a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with a probe targeted against the 5’ external region of ECSOD. DNA from C57BL/6, 129/SvEv, and BA1 (C57BL/6 x 129/SvEv) mice were used as wildtype controls. Mouse DNA genotyping R213G mice were genotyped using primers (Forward: 5’AGGCTCAAGTCTGTGCCAGAAGG-3’, Reverse: 5’TTTCCAATTCATTCACACACATGGG-3’) to distinguish wild type (357 7 bbpp amplicon) amplliconn) ffrom r knockin alleles (419 amplified eles ele es (4 4199 bbp p am amplicon). DNA was ampli lifi li f ed fi e in genoty genotyping ypi p ngg P PCR CR cocktail (10 mM primers, HiFi Buffer, mM MgSO4, minute iFi B iF uffer, 50 m M Mg M gSO O4, 10 10 mM mM DNTPs, DNTPs Ps, HiFi HiFi Taq, Taq q, water) watterr) by by a 5 m inu nute 99 99 oC hot less, 330 le 0 se econdd 995 5 oC denaturing, den de naturiing ng, 30 3 second sec econ ondd 55 55 oC annealing, an nneal alin lin i g, aand ndd 3 m minute inutte 68 inut 68 oC start for 30 cycle cycles, second extending conditions. c PCR R pr pproducts odducts were run on a 11.5% .5% 5% ag agarose garose ggel ell wi with ith a D DNA NA ladder. Human DNA NA N A genotyping t i Lungs, graciously provided by Dr. Mason (National Jewish Health), were donated through the National Disease Research Interchange (NDRI, Philadelphia, PA) and International Institute for the Advancement of Medicine (IIAM, Edison, NJ) under a Human Subject Research exempt protocol. Isolated lung DNA (Qiagen DNA tissue kit) was screened for the R213G SNP using Real-time PCR allelic discrimination assay-by-design service and the TaqMan SNP Genotyping Assay for Human SOD3 (C_2307506_10)(Applied Biosystems Life Technologies, Foster City, CA) DNA sequencing A DNA region containing the R213G SNP was sequenced using a forward primer (5’-
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AGGCTCAAGTCTG TGCCAGAAGG-3’) located in the long homology arm upstream of the R213G SNP and a reverse primer (5’-GGAACTTCGCTAGA CTAGTACGCGTG-3’) located within the Neo cassette downstream of the R213G SNP per established protocols (Molecular Research Center, National Jewish Health). DNA was amplified and the PCR product sequenced on an ABI PRISM 3100 genetic analyzer. pGEM-3zf(+) DNA was used as a positive control. EC-SOD activity assays Pulverized lung (70 mg) was homogenized in SOD activity assay buffer and total lung protein concentration was determined by BCA protein assay (Pierce Biotechnology, ology, y Rockford, IL). IL) L ___ EC-SOD was separated from intracellular SOD (SOD1 and SOD2) using ing the th he Glycoprotein Glycopprote Gl tein te in Isolation Kit, following it, ConA Con onA A (Pierce (P Pierc rcee Biotechnology) per manufacturer’s rc man anuf an u acturer’s instru instructions uct ctions with the follow minor modifications: 300 Lectin Resin the intracellular ifications: spinn columns if colu umnss contained conttainedd 30 00 ȝll ConA ConA nA L ectiin Re esiin aand nd th he in ntrraceelllul SOD fraction washing with o was on ass collected col ollect ctted d pprior rior ri ior to wa ashingg colu ccolumns. olu l mn mnss. Columns Collum umnns were wer eree inc iincubated ncuba bate ba tedd wi te ith h 3300 00 ȝl 00 ȝ elution buffer f for 15 minutes prior fer p ior to collection pr coll llectio i n off flow-through. flo l w-througgh. h Absence A sence of EC-SOD in the Ab t intracellular SOD blott while containing SOD ffraction tii was confirmed fi d by b western t bl hil hil eluted l t d fractions f tii t i i ECSOD were pooled and concentrated 10-fold using a centriprep concentrator (Amicon-ultracel10K, Millipore). Activity levels were measured by the SOD assay kit-WST (Dojindo Molecular Technologies, Maryland, USA), according to manufacturer’s instructions. Data were expressed as units of EC-SOD activity per mg of total lung protein. Heparin-sepharose binding Pooled plasma or lung tissue from ten mice (wild type or R213G) was homogenized and centrifuged; and supernatant dialyzed against 20 mM Tris –HCl pH 7.5 1. Following dialysis, the sample was applied to 1ml HiTrap Heparin Sepharose (GE Healthcare) and heparin-binding proteins eluted with a linear gradient of NaCl (0 mM±1 M) in 50 mM Tris-HCl, pH 7.5 at a flow
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rate of 1ml/min. Fractions were analyzed for EC-SOD activity by inhibition of cytochrome c reduction as described previously 2. To test if the R213G SNP altered SOD activity, we normalized activity to the relative EC-SOD content in the peak fraction. We determined the relative EC-SOD content by western blotting using a polyclonal rabbit murine EC-SOD antibody and Cy5 labeled anti-rabbit secondary antibody (Sigma). Histological analysis EC-SOD immunostaining was performed as previously described using mouse- or humanspecific antibodies. 3,4, 5. Mouse lungs were inflated-fixed at 25 cm H20 pressure with 4% % paraformaldehyde. EC-SOD KO and wild type mice were used as negative ative aand ndd posit positive itiivee ccontrols it on respectively. y y. Bronchoalveolar veo olar lavagee Lungs weree lavag lavaged ag ged wit with th 1 mL LP PBS BS S and and 200 μll of of total to otal tal bronchoalveolar b on br onch nch chooalv lveo lv eola eola l r la llavage vaage g ffluid lu uid (BALF) ((BALF BA BAL ALF was diluted in 100 mL Isoton II diluent dil iluent and andd countedd on a Z Z2 2 pa pparticle rticlle count and andd size siize analyzer analyz y er (Beckman C Co Coulter, lter lt F Fullerton, llerton ll to CA) CA). CA) Western blot analysis Homogenized lung tissue was run on a 12% Tris/Glycine SDS-PAGE gel and transferred to PVDF membrane per standard protocols 6. Blots were incubated with primary antibody as follows: Beta-actin 1:20,000 (Sigma, St. Louis, MO), EC-SOD 1:1000 (8130), 4-HNE 1:1000 (Abcam, Cambridge, MA), nitrotyrosine 1:500 (UpState, Lake Placid, NY). Following incubation with secondary antibody, blots were developed with ECL Plus (GE), imaged with STORM 860 (GE) using the fluorescent blue (430nm) setting, quantified using ImageQuant software, and expressed relative to beta-actin.
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Gene expression Total RNA was extracted using either TRIzol (Invitrogen, Carslbad CA) or RNeasy kit (Qiagen) according to the manufacturer’s instructions and differences in gene expression were determined using Real-Time RT-PCR as previously described 7. Gene expression was determined using FRPSDUDWLYHWKUHVKROGF\FOH ǻǻ&T) as suggested by the manufacturer (Applied Biosystems, Foster City, CA) normalizing each sample to 18s rRNA. The following primer sets were purchased from Assays on Demand (Applied Biosystems); 18s rRNA (cat#4310893E), SOD3 (Mm01213380-s1), SOD1 (Mm01700393_g1) and SOD2 (Mm00449726_m1). 726_m1). Proinflammatory cytokine and chemokine measurements n cchemokine nd h mok he okin i e cconcentrations oncentrations in mouse lu lun ng tissue and BALF BA ALF L were measured (ELISA (E E Cytokine and lung roraa, CO) usin ro ng eith th her the h MSD he S m SD ouusee pr roinfl flam fl am mma m to ory 7-plex x ultra a-ssens nsitiv ve kkit or TECH, Aurora, using either mouse proinflammatory 7-plex ultra-sensitive a ammat ator at oryy panel or pane nell 1 V-PLEX V PL VP EX kkit it (M Mes esoo Scale Scalle Discovery, Scal Disc Di scov sc overy, ov y Gaithersburg, Gaiith the hersb sbur sb u g, MD) ur MD) D) per per the proinflammatory (Meso r rer’s instructions. manufacturer’s tii l cell llll counts t Total differential Total BALF was centrifuged for 15 minutes at 300 X g. The cell pellet was resuspended in 200 ul PBS. 180 ul was spotted to glass slides and allowed to dry overnight. Slides were then stained for differential cell counts using HEMA 3 stain per manufacturer’s instruction (Fisher Scientific, Kalamazoo, MI). Chronic hypoxic pulmonary hypertension Four week old WT C57/BL6 and HM R213G mice were exposed to hypobaric hypoxia (395 torr) for 35 days: chambers simulated altitude (5486 m) equivalent to 10% oxygen. Normoxic control mice were maintained in Denver ambient air (1,609 m).
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Isolation of pulmonary arteries Lungs were perfused with a slurry of iron particles in warmed agarose and minced; ironcontaining PAs were separated from lung parenchyma by magnet.8 The PA was digested with collagenase to release the iron particles, and the isolated PA tissue homogenized and sonicated in RIPA buffer for western blots. Assessment of pulmonary hypertension Right ventricular systolic pressure (RVSP) was measured in anesthetized mice by direct RV puncture through a closed chest using the Cardiomax III Cardiac Output ut system (C ((Columbus olum mbu bus Instruments) as previously described 7. Right ventricular hypertrophy (RVH) (RVH H) was de ddetermined term min as ght h vventricular/left entr en tric tr i ul ulaar/left ventricular + septum um weights (RV/LV+ V+S). V+ the ratio of rig right (RV/LV+S). Assessment nt off pulmonar pulmonary ry vascular vaascullar a remodeling rem emod odelin ng poxi po xiaa-indu xi d ce du cedd mu muscul ular ul ari ar rizatio on of sma mall lll ves essse sels ls, lu ls ung ng sections sec ecti t onns were weree sstained tain tain i ed dw To evaluatee hypo hypoxia-induced muscularization small vessels, lung with PRXVHPRQRFORQDOĮ-VPRRWKPXVFOHDFWLQDQWLERG\ Į-SMA Q QRFORQDO Į-VPRRWK KPXVFOHDFWL O LQDQWLER E GG\\ Į-S SMA A )(1:100, )(1 (1:1 100 00,, Clone Cl 1A4)) and the number of positi positively siitii ell stained taiined d small ll vessels essels ls ((gly extracellular superoxide dismutase (ec-sod) in plasma is caused by a reduction of both heparin and collagen affinities. Biochem J. 2005;385:427-432. 2. Oury TD, Crapo JD, Valnickova Z, Enghild JJ. Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: A simplified, high-yield purification of extracellular superoxide dismutase. Biochem J. 1996;317( Pt 1):51-57. 3. Nozik-Grayck E, Suliman HB, Majka S, Albietz J, Van Rheen Z, Roush K, et al. Lung ec-sod overexpression attenuates hypoxic induction of egr-1 and chronic hypoxic pulmonary vascular remodeling. Am J Physiol Lung Cell Mol Physiol. 2008;295:L422-430.
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4. Bowler RP, Nicks M, Warnick K, Crapo JD. Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2002;282:L719726. 5. Oury TD, Schaefer LM, Fattman CL, Choi A, Weck KE, Watkins SC. Depletion of pulmonary ec-sod after exposure to hyperoxia. Am J Physiol Lung Cell Mol Physiol. 2002;283:L777-784. 6. Villegas LR, Kluck D, Field C, Oberley-Deegan RE, Woods C, Yeager ME, et al. Superoxide dismutase mimetic, mnte-2-pyp, attenuates chronic hypoxia-induced pulmonary hypertension, pulmonary vascular remodeling, and activation of the nalp3 inflammasome. Antioxid Redox Signal. 2013;18:1753-1764 7. Hartney JM, Brown J, Chu HW, Chang LY, Pelanda R, Torres RM. Arhgef1 regulates alpha5beta1 integrin-mediated matrix metalloproteinase expression and is required for homeostatic lung immunity. Am J Pathol. 2010;176:1157-1168. 8. Waypa GB, Chandel NS, Schumacker PT. Model for hypoxic pulmonary vasoconstriction onaryy vasoconstric icti ic tio ti involving mitochondrial oxygen sensing. Circ Res. 2001;88:1259-1266. 6 9. Lim RS, Kratzer Barry NP, Miyazaki-Anzai Kraatzer A, Bar arrry N arry P, M P, iyaz iy azak az ak ki-A Anz nzai a S,, Miyazaki Miya yaaza yaza zaki k M, M, Mantulin Mant Ma n ulin n WW, WW, W, et al. al. Multimodall cars caars microscopy microsco opy determination determi m nati t onn of ti of the th he impact im mpact ct of of diet diett on on macrophage maacrop opphagge ge infiltration innfiil il atiion and iltra lipid accumulation formation mice. Lipid 2010;51:1729-1737. mula mu lati tion on pl pplaque aq que fo orma mati ma tion iin n ap aapoe-deficient poee-ddefiiciien nt mi mice e. J L ipi p d Res. pi Rees. 2010 10;5 10 51:17729-10. Richenss TR, Linderman Horstmann SA, Lambert Xiao Keith Cigarette Lindermaan DJ DJ, Ho H rstm rs tman tm an nn SA A, La Lamb mb ber e t C, X iaoo YQ ia YQ,, Ke Keit ithh RL, et al. Ciga it a smoke impairs apoptotic cells through activation Am J a clearance off apo airs p pt p otic i cel lls th hrough ghh oxidant-dependent oxiidant-ddep pende d nt acti ivation of rhoa. A Respir Crit Care Med. Med ed. d. 20 22009;179:1011-1021. 09;1 09 ;1 179:1 79:1 79 1011011-10 01 1-10 1021 21. 21 11. Bowler RP, Barnes PJ, Crapo JD. The role of oxidative stress in chronic obstructive pulmonary disease. Copd. 2004;1:255-277. 12. Jung O, Marklund SL, Geiger H, Pedrazzini T, Busse R, Brandes RP. Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: In vivo and ex vivo evidence from ecsod-deficient mice. Circ Res. 2003;93:622-629. 13. Bowler RP, Arcaroli J, Crapo JD, Ross A, Slot JW, Abraham E. Extracellular superoxide dismutase attenuates lung injury after hemorrhage. Am J Respir Crit Care Med. 2001;164:290294. 14. Bowler RP, Arcaroli J, Abraham E, Patel M, Chang LY, Crapo JD. Evidence for extracellular superoxide dismutase as a mediator of hemorrhage-induced lung injury. Am J Physiol Lung Cell Mol Physiol. 2003;284:L680-687. 15. Bowler RP, Nicks M, Tran K, Tanner G, Chang LY, Young SK, et al. Extracellular superoxide dismutase attenuates lipopolysaccharide-induced neutrophilic inflammation. Am J Respir Cell Mol Biol. 2004;31:432-439.
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16. Tollefson AK, Oberley-Deegan RE, Butterfield KT, Nicks ME, Weaver MR, Remigio LK, et al. Endogenous enzymes (nox and ecsod) regulate smoke-induced oxidative stress. Free Radic Biol Med. 2010;49:1937-1946. 17. Yao H, Arunachalam G, Hwang JW, Chung S, Sundar IK, Kinnula VL, et al. Extracellular superoxide dismutase protects against pulmonary emphysema by attenuating oxidative fragmentation of ecm. Proc Natl Acad Sci U S A. 2010. 18. Ghio AJ, Suliman HB, Carter JD, Abushamaa AM, Folz RJ. Overexpression of extracellular superoxide dismutase decreases lung injury after exposure to oil fly ash. Am J Physiol Lung Cell Mol Physiol. 2002;283:L211-218. 19. Kang SK, Rabbani ZN, Folz RJ, Golson ML, Huang H, Yu D, et al. Overexpression of extracellular superoxide dismutase protects mice from radiation-induced lung injury. Int J Radiat Oncol Biol Phys. 2003;57:1056-1066. 20. Oury TD, Ho YS, Piantadosi CA, Crapo JD. Extracellular superoxide dismutase, nitric ide di dismut tase,, nit itri it ric oxide, ri and central nervous system o2 toxicity. Proc Natl Acad Sci U S A. 1992;89:9715-9719. 92;89::97 9715 15 971 7199 21. Hartney Venkataraman Villegas y T, Birari R, R, V e ka en kata tara ta raama mann S, S V i leega il gas L, L, Martinez Mar arrti arti tine n z M, ne M Black Black laac SM, SM, M, et et al. all Xanthine Xan anthi oxidase-derived upregulate egr-1 erk1/2 smooth muscle cells; model test r veed ros upregu rive gu ulatte egr r-1 viaa er rk1/2 inn paa smo moot othh mu ot uscle ce ellss; mode del ttoo tes de st impact im m of extracellular ullar ros in ch cchronic ronnic hhypoxia. ypooxi xia. PLoS PLoS o ONE. ON NE E. 2011;6:e27531. 20111;6 20 ;6:ee275531. 22. Xu Y, Stenmark KR, D smooth Das ass M M,, Wa W Walchak lccha h k SJ S SJ,, Ru R Ruff ff L LJ, J, D Dempsey em mpsey pssey E EC. C P C. Pulmonary ulmo ul monnary artery sm mo muscle cellss from chronicall chronically hypoxic l y hhy ll ypo p xic neonatal neonatall calves callves retain ffetal-like etal-l l like ik k andd acquire acqu q ire new gr ggrowth o properties. A Am Physiol. 1997;273:L234-245. m J Phy ysi siol ol.. 19 ol 1997 977;273 97;2 273 73:L :L23 :L23 2344 24 245. 5. 23. Juul K, Tybjaerg-Hansen A, Marklund S, Lange P, Nordestgaard BG. Genetically increased antioxidative protection and decreased chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2006;173:858-864. 24. Young RP, Hopkins R, Black PN, Eddy C, Wu L, Gamble GD, et al. Functional variants of antioxidant genes in smokers with copd and in those with normal lung function. Thorax. 2006;61:394-399. 25. Siedlinski M, van Diemen CC, Postma DS, Vonk JM, Boezen HM. Superoxide dismutases, lung function and bronchial responsiveness in a general population. Eur Respir J. 2009;33:986992. 26. Dahl M, Bowler RP, Juul K, Crapo JD, Levy S, Nordestgaard BG. Superoxide dismutase 3 polymorphism associated with reduced lung function in two large populations. Am J Respir Crit Care Med. 2008;178:906-912. 27. Sorheim IC, DeMeo DL, Washko G, Litonjua A, Sparrow D, Bowler R, et al. Polymorphisms in the superoxide dismutase-3 gene are associated with emphysema in copd. COPD. 2010;7:262-268.
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28. Marklund SL, Nilsson P, Israelsson K, Schampi I, Peltonen M, Asplund K. Two variants of extracellular-superoxide dismutase: Relationship to cardiovascular risk factors in an unselected middle-aged population. J Intern Med. 1997;242:5-14. 29. Juul K, Tybjaerg-Hansen A, Marklund S, Heegaard NH, Steffensen R, Sillesen H, et al. Genetically reduced antioxidative protection and increased ischemic heart disease risk: The copenhagen city heart study. Circulation. 2004;109:59-65. 30. Sandstrom J, Nilsson P, Karlsson K, Marklund SL. 10-fold increase in human plasma extracellular superoxide dismutase content caused by a mutation in heparin-binding domain. J Biol Chem. 1994;269:19163-19166
Figure Legends:
Figure 1. Characterization R213G SNP knockin mouse. recombination Char Ch arac a terizaati t onn off R 2133G SN NP kn nocckin mo m use. ((A) A) Homologous Homo Ho olo ogo gouus rec ecom ec ombbinaatiionn om introduces the SNP identical t identical R213G R21 2 3G G (CoG) (CoG) S NP rresulting esul es ulti ul ting ti ng iin n an n ide deenntticcal aarginine r in rg nin inee to glycine amino am m acids substitution itution in the it th he extracellular ex xtr trac accel e lu ula lar matrix-binding matr ma trix ix x-b -bin bin indi ding di ng domain dom omai aiin (ECM-BD) ain (ECM (E ECM M-BD BD)) off EC-SOD. EC-SO SOD. Bluee amino acids represent the conserved ECM-BD region and red represent R213G SNP at position 213. (B) R213G SNP homologous recombinant containing a Neo cassette (Neo). Red asterisk is site of R213G SNP. Black triangles are LoxP sites. PB ½ is probe site for Southern blots. (C) Confirmation of R213G positive clones (asterisks) by Southern blot. (D) R213G mouse DNA genotyping. Wild type (WT) mice produce a 357 bp amplicon, R213G heterozygous (HET) mice produce both a 357 bp and 419 bp amplicon, and R213G homozygous (HM) mice produce 419 bp amplicon. (E) DNA sequence confirms CoG mutation (red asterisk) changing the codon from CGG (Arginine) to GGG (Glycine). (F) Plasma EC-SOD protein of WT, HET and HM mice. (G) EC-SOD activity in units per ml of plasma (n = 6 for all genotypes). (H) Heparin sepharose binding using purified EC-SOD from wild type (WT, n = 1) open squares and dashed
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line) and R213G homozygous (R213G, n = 1) mouse (black squares and solid line) in units/ml (U/ml) of elution volume. (I) SOD activity normalized to protein (n =3 for both WT and R213G). (J) EC-SOD immunostaining in murine lung tissue. Brown staining (arrow = airway, arrowhead = vessel) for wild type (WT), R213G heterozygous (HET) and R213G homozygous (HM) mice. EC-SOD knockout mice (KO) were used as background EC-SOD staining controls. (K) EC-SOD immunostaining in mouse aorta. Brown staining (arrows) shows EC-SOD localization for WT, HET and HM mice. EC-SOD immunodepleted (ID) and Phosphate buffered saline (PBS) were used for nonspecific background staining detection. ((L) total L) EC-SOD tota al pr pprotein was analyzed in total lung tissue by western blot from WT (n = 3), HET HM ET (n = 3) aand ndd H M (n (n =3) animals. (M) EC-SOD M EC M) EC-S -SOD SOD activity act cttiv i ity in units per mg of lung lun ungg tissue protein of un of WT (n = 6), Het (n = 6) and HM (n = 55)) mice. (N) EC EC-SOD protein lung WT and R213G EC-S SOD D prote t in te n iin n lu ung tissue tisssuee fr from o W om T an nd R2 213G G human huma m n carrier. caarrr (O) EC-SOD wild R213G OD protein pro rote ro tein te i in bronchoalveolar in b on br onch ch hoa oalveo eo ola lar lavage lava vagge fluid va fluid luid (BALF) (BA B LF LF)) off w ildd type il typpe (WT) ty (WT WT) an andd R2 R R213 213 13 homozygous EC-SOD R213G u (R213G) us (R213G)) mice. (P) (P) EC E -S SOD O protein proteiin in in BALF BA ALF from from WT aand ndd R 213G human carrier caa obtained post-mortem. Data as mean r S S.E. WT. t t D t are shown h E * P < 0.05 0 05 compared d tto WT
Figure 2. Mice with R213G SNP have attenuated injury to inhaled LPS. (A) Total cells counts in BALF 0, 4 and 24 hours post-LPS. (n = 4 per group). (B) Total neutrophil counts in BALF (C) Total macrophages counts in BALF. (D) Cytokines in BALF of WT and R213G animals at 0 hours (WT n = 4, R213G n = 3), 4 hours (WT n = 8, R213G n= 4) and 24 hours (WT n = 9, R213G n = 4) post LPS inhalation. (E) 4-HNE in lung tissue. Data are presented as mean r S.E. # P < 0.05 compared to samples at 0 hours for each genotype. * P < 0.05 comparing WT to R213G samples under identical conditions.
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Figure 3. R213G SNP reduces vascular EC-SOD and increases susceptibility to pulmonary hypertension. (A) EC-SOD protein expression in pulmonary artery tissue isolated from normoxic (NX) wild type (WT) and R213G homozygous mice (R213G). (B) Right ventricular systolic pressure (RVSP mmHg) at baseline (WT n = 9, R213G n = 6) and after 35 days of chronic hypoxia (HPX) in WT (n = 8) and R213G (n = 6). (C) Right ventricular hypertrophy (RV/LV+S weights) at baseline (WT n = 8, R213G n = 6) after 35 days of HPX (WT n = 7, R213G n = 8). (D) Representative lung Į-sma immunostaining (brown signal) with counterstain by hematoxylin (blue) after 35 days of HPX. Bar = 50 microns. Arrows indicate muscularized vessels ularized small ves sse sels LGHQWLILHGE\SRVLWLYHĮ-sma immunostaining. (E) Quantitation of muscularized culariizedd small smalll vessels veesse (< 50 microns)) inn a 10x R213G after 10x 0 field fie i ld d at at baseline (WT n = 5, R2 R213 1 G n = 6) and af aft ter 35 days of HPX ((WT n = 4, R213G Collagen G n = 5). (F) Col llag gen visualized vis isualiizedd by is by TPE-SHG TPE--SHG G inn representative reeprresenttattive ve unstained unsta t in ta i ed d tissue tisssuee sections. Collagen signal), autofluorescence signal). o en ollagen n (red (red si ign gnal all), ttissue i suee au is autofl luo uore reescen ence ce ((green grree eenn sign gn nal al). l) (G) (G) Quantification Qu ifi Qu Quanti fica cati tiion of tion o collagen standardized a andardized for vessel size (n= ( = 6 for all groups). (n g oups gr p ). ) Data are shown as mean r S.E. * P < 0.01 for WT identical T compared d to R213G samples l under d id i l conditions. di i # P < 00.01 01 for f NX compared to HPX.
Figure 4. Schematic demonstrating the change in distribution of EC-SOD associated with the R213 SNP. The consequence of the change in distribution is a reduction in susceptibility to inflammation from lipopolysaccharide (LPS) and increased susceptibility to vascular disease (hypoxic pulmonary hypertension and remodeling).
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SUPPLEMENTAL MATERIAL Supplemental Methods Gene expression Total RNA was extracted using either TRIzol (Invitrogen, Carlbad, CA or RNeasy kit (Qiagen) according to the manufacturer’s instructions and differences in gene expression were determined using Real-Time RT-PCR as previously described1. Gene expression was determined using comparative threshold cycle (ΔΔCT) as suggested by the manufacturer (Applied Biosystems, Foster City, CA) normalizing each sample to 18s rRNA. The following primer sets were purchased from Assays on Demand (Applied Biosystems): 18s rRNA (cat #4310893E) and SOD3 (cat# Mm01213380-s1). Proinflammatory cytokine and chemokine measurements Cytokine and chemokine concentration in mouse lung tissue were measured using the MSD (MesoScale Discovery, Gaithersburg, MD) Proinflammatory Panel 1 V-PLEX kit. Total protein in bronchoalveolar lavage Total protein was quantitated using Coomassie Plus (Bradford) Assay kit (Thermo Fisher Scientific, Rockford, IL) per manufacturer’s instructions. Supplemental Figures
Figure legends Fig 1. (A) Expression of SOD3 was measured by Real-Time RT-PCR in whole lung tissue from WT (n = 3), HET (n = 3) and HM (n = 3) animals. Gene expression was normalized to 18s rRNA
expression and displayed relative to WT samples. (B) Expression of SOD3 was measured by Real-Time TR-PCR in isolated aortas from WT (n = 3), HET (n = 3) and HM (n = 3) animals. Gene expression was normalized to 18s rRNA expression and displayed relative to WT samples. (C) Proinflammatory cytokines and chemokines in lung tissue from WT and R213G animals at 0 (WT n = 4, R213G n = 3), 4 (WT n = 8, R213G n = 4) and 24 hours (WT n =9, R213G n = 4) post LPS inhalation. Mediators are expressed as pg per 30ug of total protein. (D) Total protein in broncoalveolar lavage fluid (BALF) in WT and R213G animals at 0 (WT n = 3, R213G n = 2), 4 (WT n = 7, R213G n = 4) and 24 hours (WT n = 4, R213G n = 7) post LPS inhalation. Data are presented as mean ± S.E. # P < 0.05 compared to samples at 0 hours for each genotype. * P < 0.05 comparing WT to R213G samples under identical conditions. Supplemental References 1.
Hartney JM, Brown J, Chu HW, Chang LY, Pelanda R, Torres RM. Arhgef1 regulates alpha5beta1 integrin-mediated matrix metalloproteinase expression and is required for homeostatic lung immunity. The American journal of pathology. 2010;176:1157-1168
Supplemental Figure 1 A
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