Oxidative stress impairs energy metabolism in primary cells and synovial tissue of patients with rheumatoid arthritis

RESEARCH ARTICLE Open Access Oxida i e s ess impai s ene gy me abolism in p ima y cells and syno ial issue o pa ien s wi h heuma oid a h i is Emese Balogh 1 , Douglas J. Veale 2 , T udy McGa y 3 , Ca l O 2 , Zol an Szekanecz 1 , Chin-Teck Ng 4,5 , U sula Fea on 3† and Monika Biniecka 2*† Abs ac Backg ound: In his s udy, we examined he e ec o oxida i e s ess on cellula ene gy me abolism and p o- angiogenic/p o-in lamma o y mechanisms o p ima y heuma oid a h i is syno ial ib oblas cells (RASFC) and human umbilical ein endo helial cells (HUVEC). Me hods: P ima y RASFC and HUVEC we e cul u ed wi h he oxida i e s ess induce 4-hyd oxy-2-nonenal (4-HNE), and ex acellula acidi ica ion a e, oxygen consump ion a e, mi ochond ial unc ion and p o-angiogenic/p o- in lamma o y mechanisms we e assessed using he Seaho se analyse , complex I–V ac i i y assays, andom mu a ion mi ochond ial cap u e assays, enzyme-linked immunoso ben assays and unc ional assays, including angiogenic ube o ma ion, mig a ion and in asion. Exp ession o angiogenic g ow h ac o s in syno ial issue (ST) was assessed by IHC in pa ien s wi h heuma oid a h i is (RA) unde going a h oscopy be o e and a e adminis a ion o umou nec osis ac o inhibi o s (TNFi). Resul s: In RASFC and HUVEC, 4-HNE-induced oxida i e s ess ep og ammed ene gy me abolism by inhibi ing mi ochond ial basal, maximal and adenosine iphospha e-linked espi a ion and ese e capaci y, coupled wi h he educed enzyma ic ac i i y o oxida i e phospho yla ion complexes III and IV. In con as , 4-HNE ele a ed basal glycolysis, glycoly ic capaci y and glycoly ic ese e, pa alleled by an inc ease in mi ochond ial DNA mu a ions and eac i e oxygen species. 4-HNE ac i a ed p o-angiogenic esponses o RASFC, which subsequen ly al e ed HUVEC in asion and mig a ion, angiogenic ube o ma ion and he elease o p o-angiogenic media o s. In i o ma ke s o angiogenesis ( ascula endo helial g ow h ac o , angiopoie in 2 [Ang2], y osine kinase ecep o [Tie2]) we e signi ican ly associa ed wi h oxida i e damage and oxygen me abolism in he in lamed syno ium. Signi ican educ ion in ST ascula i y and Ang2/Tie2 exp ession was demons a ed in pa ien s wi h RA be o e and a e adminis a ion o TNFi. Conclusions: Oxida i e s ess p omo es me abolism in a ou o glycolysis, an e ec ha may con ibu e o accele a ion o in lamma o y mechanisms and subsequen dys unc ional angiogenesis in RA. Keywo ds: Bioene ge ic me abolism, Oxida i e s ess, Angiogenesis, Rheuma oid a h i is * Co espondence: [email p o ec ed] † Equal con ibu o s 2 Cen e o A h i is and Rheuma ic Diseases, Dublin Academic Medical Cen e, S . Vincen ’s Uni e si y Hospi al, Dublin, I eland Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2018 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Balogh e al. A h i is Resea ch & The apy (2018) 20:95 h ps://doi.o g/10.1186/s13075-018-1592-1 Backg ound Angiogenesis is one o he ea lies e en s in he de- elopmen o heuma oid a h i is (RA). New blood essels in ade he syno ial memb ane, esul ing in a sel -pe pe ua ing and pe sis en in il a ion o immune cells in o he join , ans o ming he syno ial issue (ST) in o an agg essi e, umou -like ‘pannus’[1]. New capilla ies also acili a e he deli e y o su icien oxy- gen and nu ien s o suppo he p oli e a ing syno- ium. Al hough angiogenesis is a p ominen ea u e o RA, he neo ascula ne wo k is dys unc ional and ails o es o e issue oxygen homeos asis, ende ing he in lamed ST hypoxic. The inc ease in me abolic u no e o he expanding syno ial pannus ou paces he oxygen supply, esul ing in a demand o adeno- sine iphospha e (ATP) and an al e ed egula ion o cellula me abolic mechanisms [2,3]. Bioene ge ics is undamen ally impo an o all cells o enable p oli e a ion, di e en ia ion and ma u a ion, wi h mi ochond ia being cen al o biosyn he ic and bioene ge ic pa hways media ed by he ica boxylic acid (TCA) cycle. Thus, al e a ions o mi ochond ial espi - a ion can play a key ole in media ing pa hogenic mech- anisms in ch onic in lamma o y diseases [4–6]. One well-known example o mi ochond ial dys unc ion is he bioene ge ic swi ch in cell me abolism om oxida i e phospho yla ion (OXPHOS) owa ds ae obic glycolysis, known as he Wa bu g e ec . Al hough he e iciency o ATP p oduc ion pe molecule o glucose is much lowe h ough glycolysis, he yield a e is much as e han ha o OXPHOS, suppo ing apid cellula g ow h. I has been demons a ed ha he Wa bu g e ec is p esen in highly p oli e a ing and me abolically ac i e immune cells in a manne simila o ha obse ed in umou cells. In he in lamed join , an inc ease in he me abolic s a e owa ds glycolysis has been shown in p ima y heuma oid a h i is syno ial ib oblas s (RASFC), CD4 T cells, T-helpe ype 17 (T H 17) cells, mac ophages and dend i ic cells [7–10]. This is pa alleled by ele a ed lac- a e le els and diminished glucose in RA syno ial luids as well as by inc eased ac i i y o key glycoly ic enzymes in he RA syno ium, indica ing ha anae obic glycolysis is a ou ed in his hypoxic en i onmen [11–13]. Mo e ecen ly, in i o s udies by ou g oup ha e shown ha hypoxia and Toll-like ecep o 2 (TLR2)-induced in lam- ma ion p omo ed mi ochond ial dys unc ion and oxida- i e s ess and ep og ammed he na u e o cellula espi a ion in RA syno ial cells [14,15]. Oxida i e damage occu s h ough he de imen al e ec o hypoxia and is ecognised as an impo an sou ce o gen- omic ins abili y ha leads o espi a o y al e a ions. Hyp- oxia p omo es o e p oduc ion o eac i e oxygen species (ROS) ha p o oke oxida ion o polyunsa u a ed a y acids in plasma and mi ochond ial memb anes. This gene a es an a ay o p ima y lipid pe oxida ion p oduc s, which sub- sequen ly decompose and o m eac i e lipid elec ophiles, among which 4-hyd oxy-2-nonenal (4-HNE) is he mos impo an signalling molecule [16]. 4-HNE can o m co a- len adduc s wi h DNA, phospholipids and nucleophilic amino acids, impai ing hei s uc u e and biological p op- e ies. In pa icula , mi ochond ia ha e been epo ed as a p ominen a ge o 4-HNE ac i i y [17]. Mi ochond ial p o eins ela ed o mi ochond ial ene gy me abolism, such as adenosine iphospha e syn hase subuni β(ATP5B), suc- cina e dehyd ogenase la op o ein subuni and educed o m o nico inamide adenine dinucleo ide (NADH) de- hyd ogenase i on–sulphu p o ein 2 in he elec on ans- po chain (ETC), and i unc ional enzyme subuni αin he TCA cycle, a e highly suscep ible o 4-HNE-induced in- ac i a ion [18–20]. A ecen s udy has also demons a ed 4-HNE-induced inhibi ion o si uin 3, a majo mi ochon- d ial nico inamide adenine dinucleo ide (NAD + )-dependen deace ylase, wi h subsequen up- egula ion o ascula endo helial g ow h ac o (VEGF) exp ession by b eas can- ce cells [21], indica ing a close connec ion be ween oxida- i e s ess, mi ochond ial unc ion and angiogenesis. In p e ious s udies, ou g oup assessed le els o syn- o ial lipid pe oxida ion in pa ien s wi h RA and demon- s a ed a signi ican in e se co ela ion be ween 4-HNE exp ession and oxygen ension in he in lamed join , e lec ing mi ochond ial damage [22]. Subsequen ly, we ha e demons a ed ha high syno ial lipid pe oxida ion posi i ely co ela ed wi h clinical disease ac i i y sco es, and we ha e epo ed educed 4-HNE le els in pa ien s wi h RA who esponded o umou nec osis ac o (TNF) blocking he apy co esponding wi h a signi ican inc ease in pa ial oxygen p essu e in syno ial issue, in- dica ing a educ ion in syno ial oxida i e s ess as he join issue becomes less hypoxic [23]. In addi ion, i was obse ed ha inc eased syno ial in lamma ion and angiogenesis was associa ed wi h highe oxida i e s ess [22]. Gi en he impo an ole o mi ochond ial me ab- olism in he egula ion o in lamma o y and angiogenic esponses, in his s udy we in es iga ed he e ec o oxida i e s ess on he mi ochond ial bioene ge ic p o ile and he p o-angiogenic/p o-in lamma o y mechanisms in RASFC and human umbilical ein endo helial cells (HUVEC). Fu he mo e, we de e mined he e ec s o umou nec osis ac o αinhibi o s (TNFi) on he exp ession o angiogenic ma ke s in RA in ela ion o syno ial oxida i e s ess in i o. Me hods Pa ien ec ui men , a h oscopy and sample collec ion Fi een pa ien s wi h ac i e RA we e ec ui ed om he Rheuma ology Depa men o S . Vincen ’s Uni e si y Hospi al, Dublin, I eland. All pa ien s ga e ully in- o med w i en consen app o ed by he ins i u ional Balogh e al. A h i is Resea ch & The apy (2018) 20:95 Page 2 o 15 e hics commi ee, and he esea ch was pe o med in acco dance wi h he Decla a ion o Helsinki. Clinical disease ac i i y was assessed wi h he 28-join Disease Ac i i y Sco e (DAS28) using he C- eac i e p o ein le el. Unde local anaes hesia, all pa ien s wi h RA unde wen a h oscopy o he in lamed knee join p io o biologic ea men (T0) and a second a h oscopy 3 mon hs a e commencemen o TNFi (T3). ST biopsies we e used o isola ion o p ima y syno ial ib oblas s and his ological analyses. RASFC cul u e RASFC biopsies ob ained a a h oscopy we e diges ed wi h 1 mg/ml collagenase ype I (Wo hing on Biochem- ical, Lakewood, NJ, USA) in Gibco RPMI 1640 medium (The mo Fishe Scien i ic, Paisley, UK) o 4 hou s a 37 °C in humidi ied ai wi h 5% CO 2 . Dissocia ed cells we e pla ed in RPMI 1640 medium supplemen ed wi h 10% Gibco FCS (The mo Fishe Scien i ic), 20 mM 4-(2- hyd oxye hyl)-1-pipe azinee hanesul onic acid (The mo Fishe Scien i ic), penicillin (100 U/ml), s ep omycin (100 U/ml) and ampho e icin B (Fungizone 0.25 μg/ml; (In i ogen, Plymou h, MN, USA). Cells we e g own o con luence and used be ween passages 4 and 7. RASFC we e seeded on o 96-well pla es and in o T25 lasks and cul u ed in he p esence o 4-HNE (2.5 μM; Cayman Chemical, Ann A bo , MI, USA), a highly eac i e end p oduc o lipid pe oxida ion o ehicle basal medium (0.1% e hanol). The concen a ion o 4-HNE used in he expe imen s was based on a cell iabili y assay and p e- iously published s udies [24]. Following s imula ion, he e ec o ampli ied oxida i e s ess on mi ochond ial unc ion, cellula me abolism and angiogenic esponses was assessed as desc ibed below. HUVEC cul u e HUVEC (Lonza, Walke ille, MD, USA) we e incuba ed in MCDB (The mo Fishe Scien i ic) supplemen ed wi h L-glu amine (The mo Fishe Scien i ic), 0.5 ml epide mal g ow h ac o (The mo Fishe Scien i ic), 50 ml FCS (The mo Fishe Scien i ic), 0.5 ml o hyd oco isone, peni- cillin (100 U/ml; Bioscience), s ep omycin (100 U/ml; Bioscience) and Fungizone (0.25 μg/ml; Bioscience). Cells we e cul u ed a 37 °C in humidi ied ai wi h 5% CO 2 and ha es ed wi h ypsin-e hylenediamine e aace ic acid (Lonza). Cells we e used be ween passages 20 and 30. Oxygen consump ion a e and ex acellula acidi ica ion a e measu ed using Seaho se echnology Oxygen consump ion a e (OCR) and ex acellula acid- i ica ion a e (ECAR), e lec ing OXPHOS and glycolysis, espec i ely, we e measu ed be o e and a e ea men wi h oligomycin (2 μg/ml), i luo oca bonylcyanide phe- nylhyd azone (FCCP; 5 μM), an imycin A (2 μM) and 2- deoxyglucose (2-DG; 25 mM) using he Seaho se XF24 analyse (Agilen Technologies, San a Cla a, CA, USA). RASFC and HUVEC we e seeded a 30,000 cells pe well in a Seaho se XF96 cell cul u e mic opla e (Agilen Technologies) and allowed o adhe e o 24 hou s. Cells we e insed wi h assay medium (unbu e ed DMEM sup- plemen ed wi h 10 mM glucose, 1 mM sodium py u a e and 2 mM L-glu amine, pH 7.4) be o e incuba ion wi h assay medium o 30 minu es a 37 °C in a non-CO 2 in- cuba o . Following incuba ion, cells we e s imula ed wi h 4-HNE (2.5 μM) and ehicle basal medium o 2 hou s. Fou baseline OCR and ECAR measu emen s we e ob- ained o e 28 minu es be o e injec ion o speci ic me a- bolic inhibi o s. Mo eo e , o challenge he me abolic capaci y o he RASFC and HUVEC, h ee OCR and ECAR measu emen s we e ob ained o e 15 minu es ol- lowing injec ion wi h oligomycin, FCCP, an imycin A and 2-DG. In i o mi ochond ial dys unc ion and mi ochond ial DNA mu agenesis ROS p oduc ion was assessed using he DCFDA Cellula Reac i e Oxygen Species De ec ion Assay Ki (Abcam, Camb idge, UK). RASFC we e seeded in o clea -bo omed, da k-sided 96-well pla es a a densi y o 2.5 × 10 4 cells/well and allowed o a ach o e nigh . Cells we e washed in 1× bu e and s ained wi h 25 μM2′,7′-dichlo o luo escin diace a e in 1× bu e o 45 minu es a 37 °C and 5% CO 2 . A e s aining, cells we e washed, ea ed wi h 4-HNE and incuba ed a 37 °C in 5% CO 2 . ROS luo escence signal was measu ed using he Spec aMax Gemini sys em (Molecula De ices, Sunny ale, CA, USA) wi h exci a ion and emission wa eleng hs o 485 nm and 538 nm, espec i ely. Mean luo escence alues om ou wells o each condi ion we e ob ained. To cha ac e ise he equencies o andom mu a- ionsinRASFCexposed o4-HNE o 24hou s,weuseda mi ochond ial andom mu a ion cap u e assay. Mi ochond ial DNA (m DNA) was ex ac ed using a p e iously epo ed p o ocol [25]. Following ex ac ion, 10 μg o m DNA was diges ed wi h 100 U o Taq α I es ic ion enzyme (New England Biolabs, Ipswich, MA, USA), 1× bo ine se um albumin, and a Taq α I-speci ic diges ion bu e (10 mM T is HCl, 10 mM MgCl 2, 100 mM NaCl, pH 8.4) o 10 hou s, wi h 100 U o Taq α I added o he eac ion mix u e e e y hou . PCR ampli ica ion was pe o med in 25-μl eac ion mix u es con aining 12.5 μl o 2× SYBR G een B illian Mas e Mix (S a agene, La Jolla, CA, USA), 0.1 μl o u acil DNA gly- cosylase (New England Biolabs), 0.7 μl o o wa d and e- e se p ime s (10 pM/μl; In eg a ed DNA Technologies, Skokie, IL, USA), and 6.7 μlo H 2 O. The samples we e ampli ied using a Roche Ligh Cycle 480 Ins umen (Roche Diagnos ics, Indianapolis, IN, USA), acco ding o he ollowing p o ocol; 37 °C o 10 minu es, 95 °C o 10 Balogh e al. A h i is Resea ch & The apy (2018) 20:95 Page 3 o 15 minu es, ollowed by 45 cycles o 95 °C o 15 seconds and 60 °C o 1 minu e. Samples we e kep a 72 °C o 7 minu es and ollowing mel ing-cu e analysis we e imme- dia ely s o ed a −80 °C. The p ime sequences used we e as ollows: o m DNA copy numbe , 5′-ACAGTTTATG TAGCTTACCTCC-3′( o wa d) and 5′-TTGCTGCG TGCTTGATGCTTGT-3′( e e se); o andom mu a- ions, 5′-CCTCAACAGTTAAATCAACAAAACTGC-3′ ( o wa d) and 5′-GCGCTTACTTTGTAGCCTTCA-3′ ( e e se). Examina ion o mi ochond ial complexes I–V ac i i y Mi ochond ial complexes I–V OXPHOS ac i i y assay ki s (Abcam) we e used o sc een he di ec e ec o 4-HNE on all complexes o he mi ochond ial espi a- o y chain. These assays a e pe o med using whole bo ine hea mi ochond ia, a ich sou ce o OXPHOS complexes. The ac i i y o mi ochond ial complexes I–V was measu ed as pe he manu ac u e ’sins uc- ions. B ie ly, OXPHOS complex I (NADH ubiquin- one oxido educ ase) ca alyses elec on ans e om NADH o he elec on ca ie , ubiquinone, concomi- an ly pumping p o ons ac oss he inne mi ochon- d ial memb ane. The p og ession o his eac ion was moni o ed ollowing he oxida ion as a dec ease in abso bance a op ical densi y (OD) 340 nm. OXPHOS complex II (succina e-coenzyme Q educ ase) ca aly- ses elec on ans e om succina e o he elec on ca ie , ubiquinone. The p oduc , ubiquinol, is used by complex III in he espi a o y chain, and uma a e is necessa y o main ain he TCA cycle. The p oduc- ion o ubiquinol in he p esence o 4-HNE was mon- i o ed a OD 600 nm. To examine OXPHOS complex III ac i i y, succina e (elec on dono o complex II) and oxidised cy och ome c (elec on accep o o com- plex III) we e added o he mi ochond ia o s a he elec on ans e eac ion ha akes place du ing OXPHOS. The a e o coupled complex II + III eac ion was mea- su ed by moni o ing he con e sion o oxidised cy o- ch ome c in o educed o m, obse ed as an inc ease in abso bance a OD 550 nm. OXPHOS complex IV (cy o- ch ome c oxidase) ans e s elec ons om educed cy och ome c o molecula oxygen and concomi an ly pumps p o ons ac oss he inne mi ochond ial mem- b ane. The p og ession o his eac ion was moni o ed ollowing he oxida ion as a dec ease in abso bance a OD 550 nm. OXPHOS complex V makes abou 95% o a cell’s ATP using ene gy gene a ed by he p o on-mo i e o ce and can also unc ion in he e e se di ec ion in he absence o a p o on-mo i e o ce, hyd olysing ATP o gene a e adenosine diphospha e (ADP) and ino ganic phospha e. The p oduc ion o ADP by ATP syn hase can be coupled o he oxida ion o NADH o NAD + , and he p og ess o he coupled eac ion in he p esence o 4- HNE was moni o ed as a dec ease in abso bance a OD 340 nm. Resul s we e calcula ed using So Max P o 5.3 mic opla e analysis so wa e (Molecula De ices). The ac i i y o complexes I, II, IV and V is p opo ional o he dec ease in abso bance, and he linea a e o educ- ion in abso bance o e ime was calcula ed. The ac i i y o complex III is p opo ional o he inc ease in abso b- ance, and he linea a e o inc ease in abso bance o e ime was calcula ed. Fo each complex, esul s a e g aphically demons a ed as he pe cen age o enzyma ic ac i i y in he p esence o 4-HNE ela i e o he pe cen - age o basal ac i i y. Quan i ica ion o p o-angiogenic media o s in RASFC To assess he e ec s o oxida i e s ess on sec e ion o VEGF, angiopoie in 2 (Ang2), pla ele -de i ed g ow h ac o subuni B (PDGF-B), basic ib oblas g ow h ac- o (bFGF), in e leukin (IL)-8, egula ed on ac i a ion, no mal T cell exp essed and sec e ed (RANTES) and in e cellula adhesion molecule (ICAM), RASFC we e seeded in o 96-well pla es. Con luen RASFC we e se um-s a ed o 24 hou s and hen cul u ed wi h 4- HNE o 24 hou s. Supe na an s we e ha es ed, and p o ein sec e ion le els we e quan i ied using MSD as- says (Meso Scale Disco e y, Rock ille, MD, USA) o spe- ci ic enzyme-linked immunoso ben assays (ELISAs) (R&D Sys ems, Minneapolis, MN, USA). Induc ion o p o-angiogenic mechanisms o HUVEC in esponse o oxida i e s ess-ac i a ed RASFC To examine i oxida i ely ac i a ed RASFC could u he a ec p o-angiogenic mechanisms o HUVEC, RA ib o- blas cells we e s imula ed wi h 4-HNE o 24 hou s, and condi ioned media (CM) we e ha es ed. As a basal medium, we used ib oblas -condi ioned media om RASFC cul u ed in he absence o 4-HNE. Nex , he cul- u e o HUVEC was supplemen ed wi h 10% ib oblas - condi ioned media. To ensu e ha he e ec s on HUVEC unc ion we e no due o esidual 4-HNE in he 10% ib oblas -condi ioned media, HUVEC we e also cul u ed wi h RPMI 1640 medium con aining 4-HNE a he same concen a ion (0.25 μM), which is he same concen a ion as ha in he 10% RASFC CM. Following 24-hou exposu e o HUVEC o ib oblas -condi ioned media, p o-angiogenic esponses o endo helial cells we e assessed as desc ibed in he subsec ions ha ollow. HUVEC answell in asion chambe s BD BioCoa Ma igel in asion chambe s (BD Biosciences, Wokingham, UK) we e used o examine HUVEC in asion. Cells we e seeded a a densi y o 2.5 × 10 4 pe well in he mig a ion chambe on 8-μm memb anes p e-coa ed wi h Balogh e al. A h i is Resea ch & The apy (2018) 20:95 Page 4 o 15 Ma igel. HUVEC media con aining 10% ib oblas - condi ioned media was placed in he lowe well o he chambe , and cells we e allowed o mig a e o 48 hou s. Non-mig a ing HUVEC we e emo ed om he uppe su ace by gen le sc ubbing. Cells ha had in aded we e a ached o he lowe memb ane and ixed wi h 4% pa a- o maldehyde (PFA) and s ained wi h 0.1% c ys al iole . To assess he a e age numbe o in ading HUVEC, cells we e coun ed in i e andom high-powe ields. HUVEC ube o ma ion Ma igel (50 μl; BD Biosciences, San Jose, CA, USA) was pla ed in 96-well cul u e pla es a e hawing on ice and allowed o polyme ise o 30 minu es a 37 °C in humidi- ied ai wi h 5% CO 2 . HUVEC we e emo ed om cul u e, ypsinised and esuspended a a concen a ion o 4 × 10 4 cells/ml in endo helial cell g ow h medium. Fi e hund ed mic oli e s o cell suspension was added o each chambe in he p esence o 10% ib oblas -condi ioned media and cul u ed o 8 hou s. The ube analysis was de e mined om i e sequen ial ields (magni ica ion × 10) wi h a ocus on he su ace o he Ma igel by wo blinded ob- se e s and a connec ing b anch be ween wo disc e e endo helial cells was coun ed as 1 ube. HUVEC wound epai assay HUVEC we e seeded on o 24-well pla es and g own o con luence. A single sc a ch wound was induced h ough he middle o each well wi h a s e ile pipe e ip. Cells we e subsequen ly s imula ed o 24 hou s wi h 10% ib oblas - condi ioned media. HUVEC mig a ion ac oss he wound ma gins om 8 hou s was assessed and pho og aphed using a phase-con as mic oscope. Semi-quan i a i e ana- lysis o cell epopula ion o he wound was assessed. B ie ly, images o he sc a ch wound assays we e aken a × 10 magni ica ion. The mean closu e o he wound was manu- ally calcula ed om he a e age o h ee indi idual mea- su emen s om each wound. This p ocess was epea ed o all echnical eplica es. Measu emen o sc a ches a ime 0 we e designa ed as 100% open. F om his, he pe cen age o closu e o all sc a ches was calcula ed. HUVEC p oli e a ion A c ys al iole cell p oli e a ion assay was used o assess HUVEC p oli e a ion in he p esence o RASFC- condi ioned media. HUVEC we e seeded in o 96-well cul u e pla es a a densi y o 5000 cells/well and le o e nigh a 37 °C and 5% CO 2 . Nex , cells we e s imu- la ed wi h 10% ib oblas -condi ioned media o 24 hou s. Following cell cul u e, cells we e washed wi h PBS, ixed in 4% PFA and s ained wi h 1% c ys al iole solu ion. Pla es we e washed wi h ap wa e and hen d ied o e nigh . Cells we e esuspended in 1% T i on X-100 solu ion (Sigma-Ald ich, S . Louis, MO, USA), and cell numbe was measu ed wi h a mic opla e eade a a wa eleng h o 550 nm. Quan i ica ion o p o-angiogenic media o s in HUVEC HUVEC we e seeded in o 96-well pla es and le o e - nigh a 37 °C and 5% CO 2 . The ollowing day, cells we e s imula ed wi h 10% ib oblas -condi ioned media o 24 hou s. Nex , supe na an s we e ha es ed, and p o- ein sec e ion le els o Ang2 and PDGF-B we e quan i- ied by using a speci ic ELISA (R&D Sys ems). Immuno luo escence s aining o RASFC and syno ial issue Single-immuno luo escence s aining was pe o med on RASFC ollowing 24-hou cell s imula ions wi h 4-HNE. To isualise immunoexp ession o VEGF, cells we e ixed in 4% PFA and s ained wi h p ima y abbi an ibody agains VEGF (Abcam). To demons a e ST co-exp ession o ma ke s o angiogenesis, oxida i e s ess and bioene ge ics, dual-immuno luo escence s aining was pe o med on c yo- s a syno ial sec ions. ST sec ions we e ixed wi h ace one o 10 minu es and co-incuba ed wi h p ima y mouse an i- body agains human 4-HNE (GENTAUR, Kampenhou , Belgium) and wi h p ima y abbi an ibodies agains VEGF, Ang2, Tie2, ATP5B and glucose anspo e 1 (GLUT1) (all om Abcam), glyce aldehyde 3-phospha e dehyd ogenase (GAPDH) (T e igen, Gai he sbu g, MD, USA) and py u- a e kinase isozyme 2 (PKM2) (Abgen , San Diego, CA, USA). Following o e nigh incuba ion in a humidi ied chambe , RASFC and ST samples we e incuba ed wi h In i ogen Alexa Fluo 488-conjuga ed goa In i ogen Supe clonal™an i-mouse seconda y an ibody (The mo Fishe Scien i ic) and Cy™3–conjuga ed goa an i- abbi seconda y an ibody (Jackson ImmunoResea ch, Wes G o e, PA, USA) o 60 minu es and coun e s ained wi h 4′,6-diamidino-2-phenylindole (DAPI) nuclea s ain (Sigma-Ald ich) o 10 minu es. Samples we e moun ed wi h Molecula P obes an i ade moun ing medium (The mo Fishe Scien i ic) and assessed by immuno luo - escence mic oscopy (Olympus BX51; Olympus, Hambu g, Ge many). IHC and sco ing o syno ial issue IHC was pe o med using 7-μm c yos a ST sec ions and he DAKO ChemMa e EnVision ki (Dako/Agi- len Technologies, Glos up, Denma k). Sec ions we e de os ed a oom empe a u e o 20 minu es, ixed in ace one o 10 minu es and washed in PBS o 5 mi- nu es. Non-speci ic binding was blocked using 1% casein in PBS o 20 minu es. The sec ions we e incuba ed wi h abbi monoclonal p ima y an ibodies agains human VEGF, Ang2, Tie2, ATP5B (all om Abcam), GAPDH (T e igen) and mouse monoclonal an ibodies agains hu- man 4-HNE (GENTAUR). Immunoglobulin G con ol Balogh e al. A h i is Resea ch & The apy (2018) 20:95 Page 5 o 15 an ibodies we e used as nega i e con ols. Following 1- hou incuba ion wi h p ima y an ibody, endogenous pe - oxidase ac i i y was blocked using 0.3% hyd ogen pe ox- ide o 5 minu es. Slides we e incuba ed o 30 minu es wi h seconda y an ibody/ho se adish pe oxidase (Dako/ Agilen Technologies). 3,3'-Diaminobenzidine (1:50) was used o isualise s aining, and Maye ’shaema oxylin (BDH Labo a o ies, Poole, UK) was incuba ed o 30 sec- onds as a coun e s ain p io o moun ing in DPX moun - ing media. Slides we e sco ed sepa a ely o lining laye (LL), sublining laye (SL) and ascula egion (BV) using a well-es ablished and alida ed semi-quan i a i e sco ing me hod [26], whe e he pe cen age o cells ha we e posi- i e o a speci ic ma ke was compa ed wi h he pe cen - age o cells ha we e nega i e. Pe cen age posi i i y was g aded using a 0–4 scale, whe e 0 = no s ained cells, 1 = 1–25%, 2 = 25–50%, 3 = 50–75 and 4 = 75–100% s ained cells. Images we e cap u ed using an Olympus DP50 ligh mic oscope and AnalySIS so wa e (Olympus So Imaging Solu ions, Lakewood, CO, USA). S a is ical analysis IBM SPSS S a is ics e sion 20 o Windows so wa e (IBM, A monk, NY, USA) was used o s a is ical ana- lysis. Wilcoxon’s signed- ank es , Spea man’s ank- co ela ion coe icien and he Mann-Whi ney U es we e used o analysis o non-pa ame ic da a. Pa ame - ic da a we e analysed using one-way analysis o a i- ance. All p alues we e wo-sided, and p alues less han 0.05 we e conside ed s a is ically signi ican . Resul s Oxida i e s ess al e s cellula bioene ge ics in RASFC and HUVEC in i o P e ious s udies by ou g oup demons a ed al e ed cel- lula bioene ge ics in RASFC in he p esence o hypoxia [14], and we ha e also demons a ed high oxida i e s ess in he in lamed syno ium [22]. The e o e, in his s udy, we u he in es iga ed whe he oxida i e s ess in he in lamed join is in ol ed in me abolic ep og am- ming o RASFC and HUVEC. Figu e 1a demons a es ep esen a i e OCR and ECAR p o iles be o e and a e injec ions o oligomycin, FCCP, an imycin A and 2-DG in basal and 4-HNE-s imula ed RASFC. We show, o he i s ime o ou knowledge, ha inhibi ion o OCR ollowing 4-HNE-induced oxida i e s ess was associa ed wi h a shi in RASFC me abolism owa ds glycolysis. 4- HNE educed basal mi ochond ial espi a ion (p<0.05),pa - alleled by a educ ion in maximal mi ochond ial espi a ion (p< 0.001), ATP syn hesis (p= 0.1) and ese e capaci y (p< 0.01) (Fig. 1b). This me abolic ep og amming was u he accompanied by inc eased le els o basal glycolysis (p<0.01), glycoly ic capaci y (p< 0.01) and glycoly ic ese e (p=0. 2) in RASFC subjec ed o oxida i e s ess (Fig. 1b). Rep esen a i e HUVEC OCR and ECAR p o iles be- o e and a e injec ions o oligomycin, FCCP, an imy- cin A and 2-DG a e shown on Fig. 2a. Simila ly o RASFC, 4-HNE inhibi ed basal mi ochond ial espi - a ion, maximal mi ochond ial espi a ion, ATP syn hesis and ese e capaci y (all p< 0.01) wi h concomi an ele a- ion o basal glycolysis (p< 0.01) and glycoly ic ese e (p < 0.05) in HUVEC exposed o oxida i e s ess (Fig. 2b). Examina ion o mi ochond ial mu agenesis and ac i i y o enzymes o mi ochond ial OXPHOS complexes unde 4- HNE-induced oxida i e s ess We ha e p e iously shown ha inc eased m DNA mu a- ion equency and mi ochond ial dys unc ion in he RA join we e s ongly associa ed wi h syno ial in lamma ion and hypoxia [27,28]. We ha e also epo ed, a a unc- ional le el, induc ion o p o-angiogenic esponses o endo helial cells in he p esence o oxida i e s ess [29]. In he p esen s udy, we assessed he equency o m DNA mu a ions and mi ochond ial dys unc ion in RASFC sub- jec ed o 4-HNE. We obse ed inc eases in ROS p oduc- ion and m DNA poin mu a ions in RASFC in he p esence o 4-HNE compa ed wi h basal cells (p<0.001 and p= 0.06, espec i ely) (Fig. 3a). 4-HNE p o ein adduc ion may al e p o ein ac i i y; he e o e, we nex ex- amined he ac i i y o he indi idual p o eins o mi ochon- d ial OXPHOS complexes I–V. 4-HNE signi ican ly educed he ac i i y o complex III by 8% and complex IV by 70% compa ed wi h basal alues (bo h p<0.01).Lowe enzyma ic ac i i y ollowing 4-HNE s imula ion was also de ec ed o complex I by 9%, complex II by 22% and complex V by 12% (all p=0.2)(Fig.3b). In i o sec e ion o p o-angiogenic and p o-in lamma o y media o s unde oxida i e s ess condi ions Because we ound a close associa ion o edox s a e wi h ene gy me abolism in RASFC,we nex examined he e - ec o oxida i e s ess on angiogenic and in lamma o y media o s om RASFC. Figu e 4demons a es inc eased VEGF immuno luo escence s aining in RASFC cul u ed in he p esence o 4-HNE compa ed wi h he basal cells. In addi ion, 4-HNE signi ican ly inc eased sec e ion o key p o-in lamma o y and p o-angiogenic media o s compa ed wi h basal RASFC (VEGF, Ang2, bFGF, IL-8 [all p< 0.05], PDGF-B, RANTES, ICAM [all p< 0.01]). These indings, along wi h ou p e iously published in i o s udy showing TNF-α-induced mi ochond ial dys- unc ion [28], u he suppo he concep o he com- plex in e play be ween oxida i e damage, oxygen me abolism and angiogenesis in RA. The e o e, we nex de e mined angiogenic in i o esponses ollowing TNFi in 15 pa ien s wi h RA a baseline (T0) and 3 mon hs a e he commencemen o biologic ea men (T3). Addi ional ile 1: Figu e S1A shows changes o Balogh e al. A h i is Resea ch & The apy (2018) 20:95 Page 6 o 15 mac oscopic ascula i y and ST exp ession o VEGF, Ang2 and Tie2 om T0 o T3. Addi ional ile 1: Figu e S1B g aphically illus a es dec eases in ST VEGF (p=0. 1), Ang2 (p< 0.005) and Tie2 (p< 0.005) a e TNFi he apy. Oxida i e s ess-ac i a ed RASFC p omo e p o-angiogenic mechanisms in HUVEC RASFC a e known o be s ongly in ol ed in egula ing pa hological angiogenesis in he in lamed join [30]. The e o e, we nex examined i he obse ed al e a ions in cellula bioene ge ics and p o-in lamma o y p ocesses in RASFC in esponse o oxida i e s ess could subsequen ly in luencep o-angiogenicmechanismsinHUVEC.Wes im- ula ed RASFC in he p esence o absence o 4-HNE and ha - es ed he supe na an s, e med condi ioned media.Fig.5a demons a es he e ec o basal o 4-HNE RASFC-CM on in asion, he o ma ion o ube-like s uc u es and mig a ion o HUVEC. Figu e 5b g aphically illus a es ma kedly in- duced in asion (p< 0.001), p oli e a ion (p< 0.05), numbe o o med ube-like s uc u es (p< 0.001), cell mig a ion ac oss he wound (p< 0.001) and sec e ion o Ang2 and PDGF-B (bo h p alues < 0.05) in HUVEC in esponse o basal o 4-HNE RASFC-CM. To con i m ha he inc ease in p o-angiogenic e- sponses o HUVEC was due o oxida i ely ac i a ed RASFC and no o esidual 4-HNE p esen in he CM, addi ional expe imen s we e pe o med, consis ing o RPMI 1640 media supplemen ed wi h 4-HNE (0.25 μM; 4-HNE RPMI 1640 con ol), which would be a he same concen a ion o 4-HNE in he 10% RASFC-CM. A sig- ni ican inc ease in in asion (p< 0.001), numbe o o med Fig. 1 Bioene ge ic me abolism in p ima y heuma oid a h i is syno ial ib oblas cells (RASFC) subjec ed o 4-hyd oxy-2-nonenal (4-HNE)-induced oxida i e s ess. aRep esen a i e oxygen consump ion a e (OCR) and ex acellula acidi ica ion a e (ECAR) Seaho se analyse p o iles be o e and a e injec ions o oligomycin, i luo oca bonylcyanide phenylhyd azone (FCCP), an imycin A and 2-deoxyglucose (2-DG) in RASFC in he p esence and absence o 4-HNE. bBa g aphs demons a e quan i ica ion o basal mi ochond ial (M ) espi a ion, maximal M espi a ion, adenosine iphospha e (ATP) syn hesis, ese e capaci y, basal glycolysis, glycoly ic capaci y and glycoly ic ese e in RASFC (n= 5) subjec ed o oxida i e s ess. Da a a e p esen ed as mean ± SEM. *p< 0.05, **p< 0.01, and ***p< 0.001, signi ican di e ences om basal le el Balogh e al. A h i is Resea ch & The apy (2018) 20:95 Page 7 o 15 ube-like s uc u es (p< 0.01) and cell mig a ion ac oss he wound (p < 0.001) in HUVEC in esponse o 4-HNE RASFC-CM compa ed wi h 4-HNE RPMI 1640 con ol media u he suppo s he di ec e ec o 4-HNE on RASFC-induced angiogenesis in he in lamed join (Add- i ional ile 2: Figu e S2). Associa ion be ween ST angiogenesis, oxida i e s ess and bioene ge ics Finally, he co ela ion o angiogenic ac o s wi h p e i- ously assessed ma ke s o oxida i e s ess and me abol- ism in his pa ien coho was examined [14]. ST 4-HNE exp ession was associa ed wi h inc eased exp ession o VEGF ( = 0.63; p= 0.015) and Tie2 ( = 0.56; p= 0.029), GAPDH ( = 0.60; p= 0.03) and wi h educed le els o ATP5B (p=−0.52, p= 0.017). Fu he mo e, ep esen a i e immuno luo escence images demons a ing co-localisa ion o 4-HNE wi h angiogenic ac o s (VEGF, Ang2, Tie2) , as well as wi h mi ochond ial (ATP5B) and glycoly ic (GAPDH, PKM2, GLUT1) p o eins, is demons a ed in Fig. 6. Addi ional ile 3: Figu e S3 and Addi ional ile 4: Figu e S4 show single images o VEGF, Ang2, Tie2, GAPDH, PKM2, GLUT1 and ATP5B (all in ed), single images o 4-HNE immuno luo escence (in g een), as well as single images o DAPI (in blue), along wi h hei con- ols wi h iso ype-ma ched an ibodies. Discussion In his s udy, we demons a e, o he i s ime o ou knowledge, ha oxida i e s ess ep og ams cellula bio- ene ge ics o RASFC and HUVEC by down egula ing OXPHOS and p omo ing glycolysis. This change was Fig. 2 Bioene ge ic me abolism in human umbilical ein endo helial cells (HUVEC) subjec ed o 4-hyd oxy-2-nonenal (4-HNE)-induced oxida i e s ess. aRep esen a i e oxygen consump ion a e (OCR) and ex acellula acidi ica ion a e (ECAR) Seaho se analyse p o iles be o e and a e injec ions o oligomycin, i luo oca bonylcyanide phenylhyd azone (FCCP), an imycin A and 2-deoxyglucose (2-DG) in HUVEC in he p esence and absence o 4-HNE. bBa g aphs demons a e quan i ica ion o basal mi ochond ial (M ) espi a ion, maximal M espi a ion, adenosine iphospha e (ATP) syn hesis, ese e capaci y, basal glycolysis, glycoly ic capaci y and glycoly ic ese e in HUVEC (n= 3) subjec ed o oxida i e s ess. Da a a e p esen ed as mean ± SEM. *p< 0.05 and **p< 0.01, signi ican di e ences om basal le el Balogh e al. A h i is Resea ch & The apy (2018) 20:95 Page 8 o 15 e lec ed by a dec ease in mi ochond ial maximal and ATP-linked espi a ion and ese e capaci y, whe eas glycoly ic capaci y and glycoly ic ese e we e ele a ed in he p esence o 4-HNE. A bioene ge ic swi ch was coupled wi h highe ROS p oduc ion and m DNA mu a ions, in addi ion o he educed enzyma ic ac i i y o mi ochond ial complexes III and IV. Oxida i e s ess also induced sec e ion o p o-angiogenic and p o- in lamma o y media o s by RASFC. CM om 4-HNE- ac i a ed RASFC po en ia ed p o-angiogenic mecha- nisms in HUVEC, as e lec ed by ele a ed cell in asion, p oli e a ion, mig a ion, he o ma ion o ube-like s uc- u es and sec e ion o p o-angiogenic media o s. In i o co-exp ession o angiogenic ma ke s, oxida i e damage and oxygen me abolism was demons a ed in ST. Finally, a dec ease in ST angiogenesis was obse ed in pa ien s wi h RA ollowing TNFi he apy. Hypoxia is a undamen al me abolic change in ST o RA associa ed wi h ele a ed mi ochond ial ROS p oduc ion and lipid pe oxida ion. Co alen modi ica ions o m DNA, lipids and p o eins by 4-HNE ha e been epo ed o com- p omise mi ochond ial in eg i y and unc ion, including e- spi a o y me abolism, p o ein anspo a ion, mi ochond ial dynamics and quali y con ol h ough ission, usion and mi ophagy [16]. We ha e p e iously shown ha inc eased m DNA mu a ion equency and mi ochond ial dys unc- ion in he RA join co ela ed wi h g ea e hypoxia, oxida- i e s ess, ascula i y and p o-in lamma o y cy okines [27, 28]. Ou p esen in i o indings using RASFC u he demons a e high suscep ibili y o he mi ochond ial gen- ome o oxida i e damage. A mi ochond ial andom mu a- ion cap u e assay was used o quan i y he equency o andom mi ochond ial poin mu a ions in RASFC ollow- ing 4-HNE s imula ions. This me hodology elies on single- Fig. 3 Mi ochond ial mu agenesis and ac i i y o enzymes o mi ochond ial oxida i e phospho yla ion (OXPHOS) complexes unde 4-hyd oxy-2-nonenal (4-HNE)-induced oxida i e s ess. aBa g aphs demons a e inc eased p oduc ion o eac i e oxygen species (n= 7), pa alleled by he g ea e equency o mi ochond ial DNA mu a ion (n= 5) in p ima y heuma oid a h i is syno ial ib oblas cells (RASFC) in esponse o 4-HNE. bAc i i y o mi ochond ial OXPHOS complexes I–V in he p esence o 4-HNE. 4-HNE educes he ac i i y o complex I by 9%, complex II by 22%, complex III by 8%, complex IV by 70% and complex V by 12% (all complexes measu ed in iplica e). Fo each complex, esul s a e g aphically demons a ed as he pe cen age o enzyma ic ac i i y in he p esence o 4-HNE ela i e o he pe cen age o basal ac i i y. Da a is ep esen ed as Mean ± SEM, **p<0.01; ***p<0.001 signi ican ly di e en o basal Balogh e al. A h i is Resea ch & The apy (2018) 20:95 Page 9 o 15