Culture of Bovine Aortic Endothelial Cells in Galactose Media Enhances Mitochondrial Plasticity and Changes Redox Sensing, Altering Nrf2 and FOXO3 Levels

Ci a ion: Galan , L.S.; Doblado, L.; Radi, R.; de Bem, A.F.; Monsal e, M. Cul u e o Bo ine Ao ic Endo helial Cells in Galac ose Media Enhances Mi ochond ial Plas ici y and Changes Redox Sensing, Al e ing N 2 and FOXO3 Le els. An ioxidan s 2024,13, 873. h ps://doi.o g/10.3390/ an iox13070873 Academic Edi o : Claus Jacob Recei ed: 15 June 2024 Re ised: 6 July 2024 Accep ed: 16 July 2024 Published: 20 July 2024 Copy igh : © 2024 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). an ioxidan s A icle Cul u e o Bo ine Ao ic Endo helial Cells in Galac ose Media Enhances Mi ochond ial Plas ici y and Changes Redox Sensing, Al e ing N 2 and FOXO3 Le els Le icia Selinge Galan 1,2,† , Lau a Doblado 2,† , Ra ael Radi 3, And eza Fab o de Bem 1,4,5,* and Ma ia Monsal e 2,* 1Depa men o Biochemis y, Fede al Uni e si y o San a Ca a ina, Flo ianópolis 88040900, B azil; [email p o ec ed] 2 Ins i u o de In es igaciones Biomédicas Sols-Mo eale (CSIC-UAM), A u o Dupe ie 4, 28029 Mad id, Spain; [email p o ec ed] 3Depa amen o de Bioquimica y Cen o de In es igaciones Biomedicas (CEINBIO), Facul ad de Medicina, Uni e sidad de la República, Mon e ideo 2125, U uguay; [email p o ec ed] 4Depa men o Physiological Science, Ins i u e o Biological Sciences, Uni e si y o B asília, B asília 70910900, B azil 5Na ional Ins i u e o Science and Technology on Neu oimmunomodula ion, Rio de Janei o 21040360, B azil *Co espondence: [email p o ec ed] (A.F.d.B.); [email p o ec ed] (M.M.) †These au ho s con ibu ed equally o his wo k. Abs ac : Unde s anding he complex biological p ocesses o cells in cul u e, pa icula ly hose ela ed o me abolism, can be biased by cul u e condi ions, since he choice o ene gy subs a e impac s all o he main me abolic pa hways. When glucose is eplaced by galac ose, cells dec ease hei glycoly ic lux, wo king as an in i o model o limi ed nu ien a ailabili y. Howe e , he e ec o hese changes on ela ed physiological p ocesses such as edox con ol is no well documen ed, pa icula ly in endo helial cells, whe e mi ochond ial oxida ion is conside ed o be low. We e alua ed he di e ences in mi ochond ial dynamics and unc ion in endo helial cells exposed o galac ose o glucose cul u e medium. We obse ed ha cells main ained in galac ose-con aining medium show a highe mi ochond ial oxida i e capaci y, a mo e used mi ochond ial ne wo k, and highe in e cellula coupling. These ac o s a e documen ed o impac he cellula esponse o oxida i e s ess. The e o e, we analyzed he le els o wo main edox egula o s and ound ha bo ine ao ic endo helial cells (BAEC) in galac ose media had highe le els o FOXO3 and lowe le els o N 2 han hose in glucose-con aining media. Thus, cul u es o endo helial cells in a galac ose-con aining medium may p o ide a mo e sui able a ge o he s udy o in i o mi ochond ial- ela ed p ocesses han hose in glucose-con aining media; he medium deeply in luences edox signaling in hese cells. Keywo ds: endo helial cell; glucose; galac ose; mi ochond ia; an ioxidan s; oxida i e me abolism; edox; FOXO3; NRF2 1. In oduc ion The choice o ene gy subs a es o cell cul u e in i o de e mines cellula me abolic pa hways. While high glucose le els in cul u e media p omo e cell p oli e a ion, o he monosaccha ides, such as uc ose and galac ose, also se e as good ene gy sou ces, in lu- encing cell me abolism. Galac ose, o ins ance, needs o be con e ed o glucose h ough he Leloi pa hway, and i s oxida ion o py u a e does no gene a e ne ATP. The e o e, cells cul u ed wi h galac ose as he main ca bon sou ce ely on mi ochond ial oxida i e phospho yla ion (OXPHOS) o p oduce ATP, leading o a me abolic swi ch whe e glycolysis slows down and OXPHOS is ac i a ed, unlike cells cul u ed in high glucose media ha end o a o glycolysis [ 1 – 4 ]. Impo an ly, he absence o glucose and se um in he medium An ioxidan s 2024,13, 873. h ps://doi.o g/10.3390/an iox13070873 h ps://www.mdpi.com/jou nal/an ioxidan s An ioxidan s 2024,13, 873 2 o 14 can quickly dec ease cell iabili y, whe eas cells g own in he p esence o galac ose can main ain hei iabili y o e longe pe iods [5]. The essen ial ole o mi ochond ial OXPHOS in endo helial physiology is well es ab- lished [ 6 ]. Endo helial cell mi ochond ia a e highly coupled and ope a e a submaximal capaci y, showing signi ican bioene ge ic lexibili y [ 7 ]. This plas ici y is c ucial o espond- ing o s imuli such as blood low, hypoxia, angiogenesis, and in lamma ion. Addi ionally, endo helial cells exp ess PGC-1 α , a key egula o o mi ochond ial unc ion, which plays a c i ical ole in endo helial p ocesses such as p oli e a ion, mig a ion, low esponse, and ni ic oxide p oduc ion [8,9]. The o ma ion o mi ochond ial ne wo ks ha unde go usion and ission cycles in a ious cell ypes, including endo helial cells, is closely linked o mi ochond ial me abolic unc ion. Fusion is associa ed wi h “coupling e iciency” and low supe oxide p oduc- ion [ 10 ], while ission is in ol ed in emo ing damaged mi ochond ia, esponse o g ow h ac o s, and cell p oli e a ion [ 11 , 12 ]. Addi ionally, a ious s udies ha e shown ha mi- ochond ial ission se es as an adap i e esponse o cellula s ess and can impac he con ol o mi ochond ial-dependen apop osis [ 13 ]. Endo helial cells unde going oxida i e s ess exhibi mi ochond ial ission [ 14 – 16 ]. As he mi ochond ial ne wo k is c ucial o endo helial unc ion, he choice o ene gy subs a e o s udying mi ochond ial unc ion in hese cells is i al [5]. T ansc ip ion ac o s (TFs) ha espond o he in acellula edox s a us, such as FOXO1/3 and N 2, a e likely o in luence me abolic p ocesses, p omo ing he c ea ion o highly ene ge ic used mi ochond ia while main aining low le els o eac i e oxygen species (ROS) and p ese ing cell iabili y, pa icula ly in he absence o g ow h ac o s [ 17 , 18 ]. Cu en ly, ew s udies ha e conside ed hese aspec s, making i challenging o econcile cell cul u e indings wi h in i o da a on hese phenomena. The e o e, in his s udy, we aimed o e alua e he e ec s on endo helial cells’ mi ochon- d ial dynamics and unc ion o hei exposu e o a cul u e medium con aining galac ose ins ead o glucose. We ound ha hese cells ha e g ea e mi ochond ial oxida i e espi- a ion, enhanced mi ochond ial ne wo k, and inc eased in e cellula connec i i y, which is a known impo an ac o in he esponse o cells o apop o ic s imuli. In con as , cells main ained in a glucose-con aining medium display mi ochond ia agmen a ion and de- c eased oxida i e lux. In addi ion, cells main ained in a glucose-con aining medium ha e inc eased N 2 con en and unde go ime-dependen a ia ions in nuclea s. cy osolic FOXO3 localiza ion. 2. Ma e ials and Me hods Cell cul u e and ea men s—Bo ine ao ic endo helial cells (BAEC) we e ex ac ed om esh bo ine ho acic ao a as p e iously desc ibed by Pelu o and colleagues [ 9 ]. BAEC we e cul u ed in g ow h medium (DMEM) supplemen ed wi h 10% o e al bo ine se um (FBS; Gibco/In i ogen, Wal ham, MA, USA) con aining 2 mM glu amine, 100 uni s/mL penicillin, 100 µ g/mL s ep omycin, 10 mM Hepes, 25 mM glucose, and 44 mM NaHCO 3 and incuba ed a 37 ◦ C in a humidi ied a mosphe e o 5% CO 2 . Cell suspensions we e seeded in dish pla es (100 × 20 mm) in 96-, 24-, o 6-well pla es a di e en cell densi ies, depending on he expe imen al p ocedu e. Cells a passages P4–P8 we e used. When con luence eached 90%, cul u e media was changed o: (i) a glycoly ic media, DMEM (25 mM glucose) wi hou FBS o 3, 6, 12, 24, o 48 h, and (ii) an oxida i e media, glucose- ee RPMI medium wi h 5 mM galac ose and wi hou FBS o 3, 6, 12, 24, o 48 h. Fo each independen expe imen , iden ical cells, de i ed om he same o iginal cul u e dish, we e spli and hen exposed o glucose and galac ose media sepa a ely. The e o e, all he cells wi hin each independen expe imen had he same passage. Oxygen consump ion—Oxygen consump ion a es (OCR) we e measu ed using a Seaho se Bioscience sys em. To e alua e mi ochond ial oxygen consump ion in BAEC, cells we e pla ed in XF24 cell cul u e mic opla es (24-well pla es a a cell densi y o 5×103 cells/well) and submi ed o he glycoly ic (glucose) o he oxida i e (galac ose) An ioxidan s 2024,13, 873 3 o 14 p o ocol o 3, 12, o 48 h. A calib a ion ca idge (Seaho se Bioscience, Agilen Technolo- gies Spain, Las Rozas de Mad id, Spain) was equilib a ed o e nigh and hen loaded wi h unbu e ed cell cul u e media (po A), 0.6 µ M oligomycin (po B), 0.3 µ M FCCP (po C), and 0.1 µ M o enone plus 0.1 µ M an imycin A (po D), all ob ained om Sigma-Ald ich (Da ms ad , Ge many). This allowed he de e mina ion o basal espi a ion, he maximal espi a ion ese e capaci y, and ex a mi ochond ial espi a ion. The expe imen s we e un wi h he cells exposed o he same media condi ions as while in cul u e, bu unbu e ed. In all expe imen s, he p o ein concen a ion in each well was de e mined a he end o he measu emen s using he Pie ce BCA p o ein assay ki (The mo Scien i ic, Wal ham, MA, USA) a e cell lysis in RIPA bu e (Sigma-Ald ich, Da ms ad , Ge many), supplemen ed wi h a p o ease inhibi o cock ail (Comple e Mini; Roche Fa ma S.A., Mad id, Spain), and used o calib a e he oxygen consump ion da a. Mi oSOX Imaging—Mi ochond ial supe oxide was analyzed by labeling cells wi h Mi oSOX Red (Molecula P obes, Ca lsbad, CA, USA). BAEC we e g own in co e slips in 24-well cul u e (1 × 105 cells/well) pla es and submi ed o glycoly ic (glucose) o oxida i e (galac ose) p o ocols o 24 h. Then, BAEC we e incuba ed wi h 3 µ M Mi oSOX Red o 10 min, ixed wi h pa a o maldehyde, and analyzed by luo escence mic oscopy (Leica TCS SP5, Bu alo G o e, IL, USA). Immuno luo escence (IF)—BAEC we e g own on co e slips in 24-well cul u e ( 1×105 cells/well ) pla es and submi ed o he glycoly ic (glucose) o he oxida i e (galac- ose) p o ocol o 3, 6, 12, 24, o 48 h. A he end o he incuba ion pe iod, he cells we e ixed wi h 3.7% o maldehyde, pe meabilized wi h 0.1% T i on, and hen incuba ed consecu i ely wi h a p ima y an ibody di ec ed agains Tomm22 (1:200, HPA003037, MERCK, Da ms ad , Ge many) o mi ochond ial dynamics analysis and N 2 (1:200, PA1-38312, The mo Fishe , Wal ham, MA, USA) and FOXO3 (1:100, #9467, Cell signaling) edox- ela ed ansc ip ion ac o s, and hen incuba ed wi h a seconda y an ibody ( − IgG abbi ALEXA-488 conjuga e, 1:2500). Seconda y an ibody con ols a e included in Supplemen a y Figu e S1. Cells we e coun e s ained wi h DAPI, moun ed, and examined by con ocal mic oscopy (Zeiss LSM 700, Obe cochen, Ge many), as p e iously desc ibed [ 10 ]. Con ol IFs, incuba ed only wi h he seconda y an ibody, we e used o con ol o he backg ound signal ha was sub ac ed om he analysis. The o al α -Tomm22 IF signal (A las An ibodies, HPA003037, B omma, Sweden) was used o he e alua ion o he cellula mi ochond ial con en . Mi ochond ial ission was de e mined as he s anda d de ia ion o Tomm22 in ensi y signal ac oss he cell. Mi ochon- d ial subcellula dis ibu ion was also e alua ed using Tomm22 signal de e mina ion. The pe inuclea egion signal was compa ed o he o al cy osolic signal, and he asymme y o he signal in he pe inuclea egion was also e alua ed by compa ing he opposi e max and min signals in opposi e nuclea sides. The nuclei we e iden i ied by DAPI s aining. This analy ical p ocedu e has al eady been epo ed [19]. Gene exp ession analysis—Cul u ed cells we e washed wi h PBS, and o al RNA was isola ed using T izolTM eagen (The moFishe Sci., Wal ham, MA, USA) ollowing he manu ac u e ’s ins uc ions. cDNA was syn hesized om o al RNA p epa a ions by e e se ansc ip ion o 1 µ g o RNA using MMV e e se ansc ip ase (P omega Bio ech Ibé ica SL, Alcobendas, Mad id, Spain), in a inal olume o 20 µ L. The mix u e was incuba ed a 37 ◦ C o 45 min and hen cooled o 2 min a 4 ◦ C. The esul ing cDNA was used as a empla e o subsequen qPCR. The p ime s used a e lis ed below. Each 10 µ L PCR eac ion included 1 µ L cDNA, 5 µ L qPCRBIO SyG een Mas e mix (Cul ek SL, Du chche G oup, San Fe nando de Hena es, Mad id, Spain), and p ime s (0.3 µ M). Samples we e analyzed in iplica e on a Mas e cycle ® RealPlex2 (Eppendo Ibe ica SLU, San Sebas ian de los Reyes, Mad id, Spain). 36B4 was used as loading con ol. Acidic ibosomal p o ein 36B4 (36B4)-RPLP0-Gene ID: 286868 o wa d 5′-GCGACCTGGAGTCCAACTA-3′ e e se 5′-ATCTGCTGCATCTGCTTGG-3′ An ioxidan s 2024,13, 873 4 o 14 Cy och ome c (Cy c)-CYC-Gene ID: 510767 o wa d 5′-GCCAATAAGAACAAAGGCATCA-3′ e e se 5′-GTTTTGTAATAAATAAGGCAGTGG-3′ Mi ochond ial ission p o ein 1 (Fis1)-FIS1-Gene ID: 615565 o wa d 5′-GACATCCGTAAAGGCCTTGC-3′ e e se 5′-TCCATCTTTCTTCATGGCCT-3′ Mi ochond ial dynamin like GTPase (Opa1)-OPA1-Gene ID: 524142 o wa d 5′-TGGAAAATGGTACGAGAGTCAG-3′ e e se 5′-ACTGCTGAAGGATTTCTTCC-3′ Pe oxi edoxin 3 (P x3)-PRDX3 Gene ID: 281998 o wa d 5′-TTCGGGCTTCGCTCATCCGA-3′ e e se 5′-ATGGTATGAGGAACTGGTGCT-3′ ATP syn hase subuni β1 (A p5bp)-ATP5BP-Gene ID: 327675 o wa d 5′-TGTACCACCTCTTCCTGAACA-3′ e e se 5′-AGTGCCTGCTGTGACTTCTC-3′ Supe oxide dismu ase 2 (Sod2)-SOD2-Gene ID: 281496 o wa d 5′-GGAACAACAGGTCTTATCCCCCT-3′ e e se 5′-TTACTTGCTGCAAGCCGTGTATC-3′ Mi o usin 2 (M n2)-MFN2-Gene ID: 534574 o wa d 5′-TGGCGCAAGACTACAAACTG-3′ e e se 5′-TCGTCCACCAACACAGAGAG-3′ Wes e n blo ing—Whole cell ex ac s we e p epa ed as p e iously desc ibed [ 9 ]. P o ein concen a ion was e alua ed by Low y’s me hod using he RC/DC P o ein Assay (Bio-Rad). A o al o 18 µ g o p o ein ex ac was loaded on 8/15% SDS-PAGE gels, which was hen ans e ed o Immobilon-P memb anes (Cy i a, Washing on, DC, USA). P o eins o in e es we e iden i ied by wes e n blo ing using he speci ic an ibodies lis ed below, as desc ibed in [ 9 ]. The p o eins we e isualized using Cla i y ECL Subs a e (BioRad Spain, Alcobendas, Mad id, Spain), and he image was cap u ed wi h a chemiluminescence memb ane’s eade (Ni co SL, Mos oles, Mad id, Spain). The p o ein bands we e quan i ied using ImageJ so wa e. O iginal scanned blo s a e included in he Supplemen a y Ma e ials. Tubulin was used as a loading con ol. α-OPA1 1:500 Sigma-Ald ich, Da ms ad , Ge many Re . HPA036926 α-Tomm22 1:2000 A las An ibodies, B omma, Sweden Re . HPA003037 α-PGC-1 α1:1000 Cayman Chemical, Ann A bo , MI, USA Re . 101707 α-Tubulin 1:1000 Sigma-Ald ich, Da ms ad , Ge many Re . 9026 Image analysis—ImageJ so wa e was used o he analysis o a eas, signals in an a ea, and c oss-sec ion signals om luo escence and con ocal mic oscopic images o Tomm22, N 2, FOXO3, and Mi oSOX and om wes e n blo bands o α -Tubulin, α -Tomm22, and α-OPA1. S a is ics—S a is ical analysis and g aphics we e made using G aphPad PRISM ® so - wa e e sion 9 o Windows (G aphPad So wa e, San Diego, CA, USA). No mal (Gaussian) dis ibu ion was e alua ed wi h he Shapi o–Wilk no mali y es . Signi ican di e ences among g oups we e e alua ed by unpai ed - es o wo-way analysis o a iance (ANOVA), depending on he expe imen al design. Mul iple compa isons we e pe o med using Bon- e oni’s pos -hoc es . Resul s a e exp essed as mean ± SEM. p< 0.05 was conside ed s a is ically signi ican . n ≥ 4 in all expe imen s. The numbe o independen expe imen s o each pa icula expe imen is indica ed in he co esponding igu e legend. An ioxidan s 2024,13, 873 5 o 14 3. Resul s 3.1. E ec o Glucose- o Galac ose-Con aining Medium in Cellula Respi a ion and Mi ochond ial Supe oxide Radical (O2•−) Le els in BAEC Mi ochond ial oxygen consump ion in BAEC condi ioned in glucose o galac ose medium is depic ed in Figu e 1. Using a Seaho se sys em, he mi ochond ial oxygen con- sump ion in BAEC was e alua ed. BAEC a e p ima y cell cul u es, and cellula espi a ion can be a ec ed by cell passage. In ac , cells wi h passage 4 (P4) displayed highe basal espi a ion a es, as well as a highe maximum espi a o y ese e capaci y, es ima ed ol- lowing FCCP s imula ion, han hose in passages 5 (P5) o 8 (P8) (Supplemen a y Figu e S2). The e o e, i is e y impo an o es he cellula espi a ion– ela ed pa hways using ea ly cell passages o ob ain obus esul s in p ima y endo helial cell cul u es. In subsequen expe imen s wi h BAEC, we used P4–P8 passages, as indica ed in he igu e legends. In o de o con i m he ac i a ion o oxida i e phospho yla ion in a galac ose-con aining medium, we compa ed he mi ochond ial oxygen consump ion a es in BAEC condi ioned in galac ose and glucose cell cul u e media (in se um dep i a ion condi ions) a 3, 12, and 48 h (Figu e 1A,B). In he absence o se um, he maximum espi a o y ese e capaci y a e 12–48 h o incuba ion o BAEC condi ioned in cul u e medium con aining galac ose was ound o be signi ican ly highe when compa ed o ha obse ed in cells condi ioned in cul u e medium con aining glucose. Toge he , hese da a sugges ha galac ose inc eased he maximal oxida i e capaci y o BAEC and possibly also i s me abolic plas ici y. An ioxidan s 2024, 13, x FOR PEER REVIEW 5 o 14 independen expe imen s o each pa icula expe imen is indica ed in he co esponding igu e legend. 3. Resul s 3.1. Effec o Glucose- o Galac ose-Con aining Medium in Cellula Respi a ion and Mi ochond ial Supe oxide Radical (O2•−) Le els in BAEC Mi ochond ial oxygen consump ion in BAEC condi ioned in glucose o galac ose medium is depic ed in Figu e 1. Using a Seaho se sys em, he mi ochond ial oxygen consump ion in BAEC was e alua ed. BAEC a e p ima y cell cul u es, and cellula espi a ion can be affec ed by cell passage. In ac , cells wi h passage 4 (P4) displayed highe basal espi a ion a es, as well as a highe maximum espi a o y ese e capaci y, es ima ed ollowing FCCP s imula ion, han hose in passages 5 (P5) o 8 (P8) (Supplemen a y Figu e S2). The e o e, i is e y impo an o es he cellula espi a ion– ela ed pa hways using ea ly cell passages o ob ain obus esul s in p ima y endo helial cell cul u es. In subsequen expe imen s wi h BAEC, we used P4–P8 passages, as indica ed in he igu e legends. In o de o con i m he ac i a ion o oxida i e phospho yla ion in a galac ose-con aining medium, we compa ed he mi ochond ial oxygen consump ion a es in BAEC condi ioned in galac ose and glucose cell cul u e media (in se um dep i a ion condi ions) a 3, 12, and 48 h (Figu e 1A,B). In he absence o se um, he maximum espi a o y ese e capaci y a e 12–48 h o incuba ion o BAEC condi ioned in cul u e medium con aining galac ose was ound o be signi ican ly highe when compa ed o ha obse ed in cells condi ioned in cul u e medium con aining glucose. Toge he , hese da a sugges ha galac ose inc eased he maximal oxida i e capaci y o BAEC and possibly also i s me abolic plas ici y. Figu e 1. Effec o glucose- o galac ose-con aining medium (wi hou FBS) on mi ochond ial espi a ion and mi ochond ial O2− p oduc ion in BAEC (P4–P6). (A) Rep esen a i e espi ome y assay o BAEC condi ioned in cul u e media con aining glucose (Glu) o galac ose (Gal) o 12 h. A ows indica e he ime o adding he oligomycin, FCCP, and o enone plus an imycin. (B) Maximum espi a o y ese e capaci y: O2 consump ion a e he addi ion o FCCP. Each independen expe imen was pe o med using cells in he same cell passage exposed o glucose o galac ose media. O2 consump ion a es we e co ec ed o p o ein concen a ion. Da a we e ep esen ed as mean ± SEM (n = 4). * p < 0.05 indica es s a is ical diffe ence be ween he g oups (glucose s. galac ose), by wo-way ANOVA ollowed by Bon e oni’s pos -hoc es . Da a we e ep esen ed as mean ± SEM (n = 4). * p < 0.05 indica es s a is ical diffe ence be ween he g oups (glucose s. galac ose), by unpai ed - es . Mi ochond ia a e majo modula o s o cellula eac i e species (RS). Thus, o es he impac o he media con aining glucose o galac ose on mi ochond ial RS, we incuba ed BAEC wi h Mi oSOX Red, a eagen ha labels mi ochond ial supe oxide. We did no de ec diffe ences in mi ochond ial O2•− le el when BAEC we e cul i a ed in media Figu e 1. E ec o glucose- o galac ose-con aining medium (wi hou FBS) on mi ochond ial espi a- ion and mi ochond ial O 2− p oduc ion in BAEC (P4–P6). (A) Rep esen a i e espi ome y assay o BAEC condi ioned in cul u e media con aining glucose (Glu) o galac ose (Gal) o 12 h. A ows indica e he ime o adding he oligomycin, FCCP, and o enone plus an imycin. (B) Maximum espi a o y ese e capaci y: O 2 consump ion a e he addi ion o FCCP. Each independen expe i- men was pe o med using cells in he same cell passage exposed o glucose o galac ose media. O 2 consump ion a es we e co ec ed o p o ein concen a ion. Da a we e ep esen ed as mean ±SEM ( n=4 ). * p< 0.05 indica es s a is ical di e ence be ween he g oups (glucose s. galac ose), by wo- way ANOVA ollowed by Bon e oni’s pos -hoc es . Da a we e ep esen ed as mean ± SEM ( n=4 ). *p< 0.05 indica es s a is ical di e ence be ween he g oups (glucose s. galac ose), by unpai ed - es . Mi ochond ia a e majo modula o s o cellula eac i e species (RS). Thus, o es he impac o he media con aining glucose o galac ose on mi ochond ial RS, we incuba ed BAEC wi h Mi oSOX Red, a eagen ha labels mi ochond ial supe oxide. We did no de ec di e ences in mi ochond ial O 2•− le el when BAEC we e cul i a ed in media con aining glucose o galac ose (Supplemen a y Figu e S2), sugges ing ha he edox balance was no signi ican ly a ec ed by medium manipula ions. 3.2. Mi ochond ial Dynamics o BAEC Condi ioned in Glucose o Galac ose Medium Mi ochond ia a e plas ic o ganelles ha equen ly change hei mo phology, olume, and in acellula dis ibu ion in esponse o luc ua ions in me abolic demands. Thus, he dis up ion o his balance esul s in mi ochond ial dys unc ion. Tomm22 is pa o a p o ein An ioxidan s 2024,13, 873 6 o 14 anslocase complex ound in he ou e mi ochond ial memb ane and which is commonly used as a ma ke o mi ochond ial olume, mo phology, and in acellula mo emen by immuno luo escence. As shown in Figu e 2A,B, a e 3, 6, 12, 24, o 48 h in a medium con aining glucose o galac ose in he absence o FBS, no change in mi ochond ial o al olume con en was obse ed, sugges ing ha he obse ed inc ease in oxida i e capaci y in galac ose media was no ela ed o inc eases in mi ochond ial mass. This obse a ion was suppo ed by wes e n blo analysis o Tomm22 cellula con en a 24 h; a simila esul was also ob ained o ano he mi ochond ial p o ein, OPA1, a ma ke o c is ae densi y, and he a io o he wo was also p ese ed in he wo media (Supplemen a y Figu e S4A). Fu he mo e, we did no de ec signi ican gene exp ession changes in genes coding o mi ochond ial p o eins, including componen s o he elec on anspo chain/ETC (Cy c, A p5bp), an ioxidan s (Sod2, P x3), and egula o s o mi ochond ial dynamics (Mn 2,Opa1, Fis1) a 24 h, al hough a gene al endency o highe alues could be obse ed o cells in galac ose (Supplemen a y Figu e S4B). Ne e heless, we obse ed ha he mi ochond ia o BAEC in galac ose we e signi - ican ly mo e used han hose in glucose a 12 and 24 h (Figu e 2C). I was also no ed ha he s a us o mi ochond ia ission in BAEC exposed o galac ose was s able o e ime (Figu e 2C), sugges ing ha se um dep i a ion induced mi ochond ial ission only when cells we e in glucose media. Changes in he in acellula dis ibu ion o mi ochond ia we e also e alua ed, since mi ochond ial unc ion is also dependen on i s subcellula localiza ion by he de e mina ion o pe inuclea / o al and le / igh nuclea a ios, bu no signi ican di e ences we e iden i ied in he cellula esponse o cul u e medium o ime o condi ioning (Supplemen a y Figu e S5A,B). Nex , we e alua ed he in e cellula coo dina ion o he mi ochond ial ne wo k, since in e cellula con ac s p o ide me abolic coupling ha allows coo dina ed cellula esponses wi hin issues ha a e o ele ance in he esponse o s imuli (i.e., oxida i e s ess, in lam- ma ion, and apop osis). The e o e, we decided o e alua e he in e cellula a iabili y (s anda d de ia ion) o he immuno luo escence signal o Tomm22 as a su oga e ma ke o in e cellula me abolic coupling. We obse ed ha cells main ained in he galac ose medium displayed signi ican ly lowe in e cellula a iabili y o Tomm22 signal han cells in he glucose medium (Figu e 2D,E and Supplemen a y Figu e S5C,D). These esul s could be indica i e o a mo e s able/ex ended in e cellula con ac su ace ha would esul in a mo e coo dina ed in e cellula esponse in galac ose- ea ed BAEC han in BAEC main ained in a glucose medium. 3.3. E ec o Glucose- o Galac ose-Con aining Medium on T ansloca ion and Exp ession o N 2 In o de o e alua e how subs a e u iliza ion impac ed on he endo helial cell capaci y o espond o oxida i e s ess s imuli, we e alua ed he basal le els and subcellula local- iza ion o he main edox-sensi i e ansc ip ion ac o s, N 2 and FOXO3, ha ansloca e o he nuclei ollowing oxida i e s imula ion. We i s used immuno luo escence (IF) o in es iga e N 2 le els and subcellula localiza ion (Figu e 3A). E alua ing he signal in- ensi y o cy osolic (Figu e 3B) and nuclea (Figu e 3C) N 2, we obse ed a signi ican ly highe o al le el o N 2 in cells main ained in a medium con aining glucose han in cells main ained in a medium con aining galac ose a all imes es ed, and his di e ence was ob- se ed bo h in he cy osol and he nucleus. Ne e heless, when he N 2 nuclea /cy osolic a io was e alua ed, i was no ed ha a 48 h BAEC in galac ose had a highe a io han BAEC in glucose (Supplemen a y Figu e S6A). These esul s sugges ha o main ain ROS homeos asis, BAEC in glucose need highe le els o N 2, bo h in he nucleus and in he cy osol, han BAEC main ained in a galac ose medium. An ioxidan s 2024,13, 873 7 o 14 An ioxidan s 2024, 13, x FOR PEER REVIEW 7 o 14 Figu e 2. Effec o glucose- o galac ose-con aining medium on mi ochond ial dynamic and in e cellula a iabili y in BAEC (P4-6). (A) Rep esen a i e image o immuno luo escence om Tomm22 (g een) and DAPI (blue) in BAEC o 24 h; he whi e ba s ep esen 50 µm. (B) Quan i ica ion o mi ochond ial o al o (C) Mi ochond ial ission. In e cellula a iabili y was pe o med by s anda d de ia ion o (D) mi ochond ial o al and (E) mi ochond ial ission. Each independen expe imen was pe o med using cells in he same cell passage exposed o glucose o galac ose media. Da a in g aphs ep esen mean ± SEM (n = 4). * p < 0.05, ** p < 0.01 indica es a s a is ical diffe ence be ween he g oups o glucose (Glu o dashed line) and galac ose (Gal o black ba s), # p < 0.05, ## p < 0.01 indica es ime-dependen s a is ical diffe ence be ween he esponses o Glu and Gal by wo-way ANOVA, ollowed by Bon e oni’s pos -hoc es . 3.3. Effec o Glucose- o Galac ose-Con aining Medium on T ansloca ion and Exp ession o N 2 In o de o e alua e how subs a e u iliza ion impac ed on he endo helial cell capaci y o espond o oxida i e s ess s imuli, we e alua ed he basal le els and subcellula localiza ion o he main edox-sensi i e ansc ip ion ac o s, N 2 and Figu e 2. E ec o glucose- o galac ose-con aining medium on mi ochond ial dynamic and in e cel- lula a iabili y in BAEC (P4-6). (A) Rep esen a i e image o immuno luo escence om Tomm22 (g een) and DAPI (blue) in BAEC o 24 h; he whi e ba s ep esen 50 µ m. (B) Quan i ica ion o mi ochond ial o al o (C) Mi ochond ial ission. In e cellula a iabili y was pe o med by s anda d de ia ion o (D) mi ochond ial o al and (E) mi ochond ial ission. Each independen expe imen was pe o med using cells in he same cell passage exposed o glucose o galac ose media. Da a in g aphs ep esen mean ± SEM (n = 4). * p< 0.05, ** p< 0.01 indica es a s a is ical di e ence be ween he g oups o glucose (Glu o dashed line) and galac ose (Gal o black ba s), # p< 0.05, ## p< 0.01 indica es ime-dependen s a is ical di e ence be ween he esponses o Glu and Gal by wo-way ANOVA, ollowed by Bon e oni’s pos -hoc es . An ioxidan s 2024,13, 873 8 o 14 An ioxidan s 2024, 13, x FOR PEER REVIEW 8 o 14 FOXO3, ha ansloca e o he nuclei ollowing oxida i e s imula ion. We i s used im- muno luo escence (IF) o in es iga e N 2 le els and subcellula localiza ion (Figu e 3A). E alua ing he signal in ensi y o cy osolic (Figu e 3B) and nuclea (Figu e 3C) N 2, we obse ed a signi ican ly highe o al le el o N 2 in cells main ained in a medium con- aining glucose han in cells main ained in a medium con aining galac ose a all imes es ed, and his diffe ence was obse ed bo h in he cy osol and he nucleus. Ne e heless, when he N 2 nuclea /cy osolic a io was e alua ed, i was no ed ha a 48 h BAEC in galac ose had a highe a io han BAEC in glucose (Supplemen a y Figu e S6A). These esul s sugges ha o main ain ROS homeos asis, BAEC in glucose need highe le els o N 2, bo h in he nucleus and in he cy osol, han BAEC main ained in a galac ose me- dium. Figu e 3. Effec o glucose- o galac ose-con aining medium on N 2 exp ession in BAEC (P4-6). (A) Rep esen a i e image o immuno luo escence om N 2 (g een) and DAPI (blue) in BAEC a e 24 h o incuba ion wi h glucose o galac ose; whi e ba s ep esen 50 µm. (B) In ensi y o cy osolic N 2, (C) in ensi y o nuclea N 2. Each independen expe imen was pe o med using cells in he same cell passage exposed o glucose o galac ose media. Da a we e ep esen ed as mean ± SEM (n = 4). * p < 0.05 indica es s a is ical diffe ence be ween glucose (Glu) and galac ose (Gal) condi ions by wo- way ANOVA, ollowed by Bon e oni’s pos -hoc es . 3.4. Effec o Glucose- o Galac ose-Con aining Medium on T ansloca ion and Exp ession o FOXO3 T ansc ip ion Fac o Nex , we e alua ed by IF he signal in ensi y o cy osolic (Figu e 4B) and nuclea (Figu e 4C) FOXO3, as shown in Figu e 4. In con as wi h he N 2 da a, a 24 h and 48 h o incuba ion, we obse ed a highe o al le el o cy osolic FOXO3 in BAEC main ained in a galac ose medium. Consis en ly, a 48 h o incuba ion, he le els o nuclea FOXO3 we e signi ican ly highe in BAEC main ained a galac ose-con aining medium. Fu he mo e, i was no ed ha BAEC in glucose showed a signi ican ime-dependen educ ion in Figu e 3. E ec o glucose- o galac ose-con aining medium on N 2 exp ession in BAEC (P4-6). (A) Rep esen a i e image o immuno luo escence om N 2 (g een) and DAPI (blue) in BAEC a e 24 h o incuba ion wi h glucose o galac ose; whi e ba s ep esen 50 µ m. (B) In ensi y o cy osolic N 2, (C) in ensi y o nuclea N 2. Each independen expe imen was pe o med using cells in he same cell passage exposed o glucose o galac ose media. Da a we e ep esen ed as mean ± SEM (n = 4 ). * p< 0.05 indica es s a is ical di e ence be ween glucose (Glu) and galac ose (Gal) condi ions by wo-way ANOVA, ollowed by Bon e oni’s pos -hoc es . 3.4. E ec o Glucose- o Galac ose-Con aining Medium on T ansloca ion and Exp ession o FOXO3 T ansc ip ion Fac o Nex , we e alua ed by IF he signal in ensi y o cy osolic (Figu e 4B) and nuclea (Figu e 4C) FOXO3, as shown in Figu e 4. In con as wi h he N 2 da a, a 24 h and 48 h o incuba ion, we obse ed a highe o al le el o cy osolic FOXO3 in BAEC main ained in a galac ose medium. Consis en ly, a 48 h o incuba ion, he le els o nuclea FOXO3 we e signi ican ly highe in BAEC main ained a galac ose-con aining medium. Fu he mo e, i was no ed ha BAEC in glucose showed a signi ican ime-dependen educ ion in FOXO3. Howe e , no signi ican di e ence in he nuclea /cy osol a io was obse ed be ween in BAEC in glucose and galac ose media. (Supplemen a y Figu e S6B). These esul s sugges ha galac ose- ea ed cells may be mo e dependen on FOXO3 o main ain edox homeos asis han glucose- ea ed cells. An ioxidan s 2024,13, 873 9 o 14 An ioxidan s 2024, 13, x FOR PEER REVIEW 9 o 14 FOXO3. Howe e , no signi ican diffe ence in he nuclea /cy osol a io was obse ed be- ween in BAEC in glucose and galac ose media. (Supplemen a y Figu e S6B). These esul s sugges ha galac ose- ea ed cells may be mo e dependen on FOXO3 o main ain edox homeos asis han glucose- ea ed cells. Figu e 4. Effec o glucose- o galac ose-con aining medium on FOXO3 exp ession in BAEC (P4–6). (A) Rep esen a i e image o IF om FOXO3 (g een) and DAPI (blue) in BAEC a e 24 h o incuba- ion wi h glucose o galac ose; whi e scale ba s ep esen 50 µm. (B) In ensi y o cy osolic FOXO3, (C) in ensi y o nuclea FOXO3. Each independen expe imen was pe o med using cells in he same cell passage exposed o glucose o galac ose media. Da a we e ep esen ed as mean ± SEM (n = 4). * p < 0.05 indica es s a is ical diffe ence be ween glucose (Glu) and galac ose (Gal) condi ions by wo-way ANOVA, ollowed by Bon e oni’s pos -hoc es . 4. Discussion The esul s p esen ed in his s udy show ha p ima y endo helial cells effec i ely modula e hei me abolism in esponse o cell cul u e condi ions, which signi ican ly im- pac s on he mechanisms ha egula e cellula edox homeos asis. The e o e, he use o cul u e media con aining galac ose ins ead o glucose is pe inen o he s udy o endo- helial physiological p ocesses in ol ing mi ochond ial unc ion and/o edox con ol, such as angiogenesis, NO p oduc ion, esponse o blood low, in e ac ion wi h immune cells, in e cellula communica ion, and pe meabili y. Addi ionally, we no ed ha cells in galac ose exhibi ed highe oxida i e capaci y, mo e used mi ochond ia, and seem o be mo e me abolically coupled h ough in e cellula communica ion han BAEC in glucose media. Fu he mo e, we obse ed ha cells in glucose ha e highe le els o N 2, while hose in galac ose ha e highe le els o FOXO3, sugges ing ha he mechanisms in ol ed in RS con ol a e signi ican ly al e ed by cul u e media condi ions. Figu e 4. E ec o glucose- o galac ose-con aining medium on FOXO3 exp ession in BAEC ( P4–6 ). (A) Rep esen a i e image o IF om FOXO3 (g een) and DAPI (blue) in BAEC a e 24 h o incuba ion wi h glucose o galac ose; whi e scale ba s ep esen 50 µ m. (B) In ensi y o cy osolic FOXO3, (C) in ensi y o nuclea FOXO3. Each independen expe imen was pe o med using cells in he same cell passage exposed o glucose o galac ose media. Da a we e ep esen ed as mean ± SEM ( n=4 ). *p< 0.05 indica es s a is ical di e ence be ween glucose (Glu) and galac ose (Gal) condi ions by wo-way ANOVA, ollowed by Bon e oni’s pos -hoc es . 4. Discussion The esul s p esen ed in his s udy show ha p ima y endo helial cells e ec i ely modula e hei me abolism in esponse o cell cul u e condi ions, which signi ican ly impac s on he mechanisms ha egula e cellula edox homeos asis. The e o e, he use o cul u e media con aining galac ose ins ead o glucose is pe inen o he s udy o endo helial physiological p ocesses in ol ing mi ochond ial unc ion and/o edox con ol, such as angiogenesis, NO p oduc ion, esponse o blood low, in e ac ion wi h immune cells, in e cellula communica ion, and pe meabili y. Addi ionally, we no ed ha cells in galac ose exhibi ed highe oxida i e capaci y, mo e used mi ochond ia, and seem o be mo e me abolically coupled h ough in e cellula communica ion han BAEC in glucose media. Fu he mo e, we obse ed ha cells in glucose ha e highe le els o N 2, while hose in galac ose ha e highe le els o FOXO3, sugges ing ha he mechanisms in ol ed in RS con ol a e signi ican ly al e ed by cul u e media condi ions. While he e is widesp ead e idence showcasing he unc ional ele ance o mi ochon- d ia in endo helial physiology and edox homeos asis, his aspec is o en o e looked [ 20 ]. Due o he so-called C ab ee e ec , well documen ed in he li e a u e o di e en cell ypes, BAEC in glucose media unde se um dep i a ion condi ions exhibi ed lowe maximal OCRs han cells main ained in galac ose-con aining media, sugges ing an enhanced me abolic plas ici y. Simila ly, inc eased oxida i e me abolism and dec eased anae obic me abolism