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
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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