A icle
Compe i ion o endo helial cell pola i y d i es
ascula mo phogenesis in he mouse e ina
G aphical abs ac
Highligh s
dEC pola i y pa e ns de ine he sp ou ing o emodeling (S>R)
ansi ion
dBlood low and VEGFA compe e o es ablish EC pola iza ion
pa e ns in he mouse e ina
dBlood low and VEGFA egula e EC pola i y using dis inc
mechanosensi i e complexes
Au ho s
Ped o Ba bacena,
Ma ia Dominguez-Cejudo,
Ca a ina G. Fonseca, ...,
Anne Eichmann, Miguel O. Be nabeu,
Cla
´udio A. F anco
Co espondence
c [email protected]
In b ie
Ba bacena and Dominguez-Cejudo e al.
demons a e ha shea s ess and
VEGFA compe e o es ablish he
pola iza ion axis o endo helial cells,
using he mouse e ina as a model. This
compe i ion egula es he ansi ion om
he sp ou ing angiogenesis p og am
owa d ascula emodeling, hus
p omo ing signi ican mo phological and
unc ional changes in blood essels.
Ba bacena e al., 2022, De elopmen al Cell 57, 2321–2333
Oc obe 10, 2022 ª2022 The Au ho (s). Published by Else ie Inc.
h ps://doi.o g/10.1016/j.de cel.2022.09.002 ll
A icle
Compe i ion o endo helial cell
pola i y d i es ascula mo phogenesis
in he mouse e ina
Ped o Ba bacena,
1,14
Ma ia Dominguez-Cejudo,
1,14
Ca a ina G. Fonseca,
1
Manuel Go
´mez-Gonza
´lez,
2
Lau a M. Fau e,
2
Geo gia Za kada,
3
And eia Pena,
1
Anna Pezza ossa,
1,4
Daniela Ramalho,
1
Ylenia Gia a ano,
5
Ma ie Oua ne
´,
1
Da id Ba a a,
1
Isabela C. Fo una o,
1
Lenka Henao Misiko a,
1
Ian Mauldin,
5,6
Yulia Ca alho,
1
Xa ie T epa ,
2,7,8,9
Pe e Roca-Cusachs,
2,8
Anne Eichmann,
3,10,11
Miguel O. Be nabeu,
5,12
and Cla
´udio A. F anco
1,13,15,
*
1
Ins i u o de Medicina Molecula Joa
˜o Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa, Lisbon, Po ugal
2
Ins i u e o Bioenginee ing o Ca alonia (IBEC), he Ba celona Ins i u e o Technology (BIST), Ba celona, Spain
3
Ca dio ascula Resea ch Cen e , Depa men o In e nal Medicine, Yale Uni e si y School o Medicine, New Ha en, CT, USA
4
Champalimaud Founda ion, Champalimaud Resea ch, Lisbon, Po ugal
5
Cen e o Medical In o ma ics, Ushe Ins i u e, The Uni e si y o Edinbu gh, Edinbu gh, UK
6
School o In o ma ics, The Uni e si y o Edinbu gh, Edinbu gh, UK
7
Facul a de Medicina, Uni e si a de Ba celona, Ba celona, Spain
8
Ins i ucio
´Ca alana de Rece ca i Es udis A anc¸ a s (ICREA), Ba celona, Spain
9
Cen o de In es igacio
´n Biome
´dica en Red en Bioingenie ı
´a, Bioma e iales y Nanomedicina (CIBER-BBN), Ba celona, Spain
10
Depa men o Cellula and Molecula Physiology, Yale Uni e si y School o Medicine, New Ha en, CT, USA
11
Uni e si e
´de Pa is, PARCC, INSERM, 75006 Pa is, F ance
12
The Bayes Cen e, The Uni e si y o Edinbu gh, Edinbu gh, UK
13
Uni e sidade Ca o
´lica Po uguesa, Ca o
´lica Medical School, Ca o
´lica Biomedical Resea ch Cen e, Lisbon, Po ugal
14
These au ho s con ibu ed equally
15
Lead con ac
*Co espondence: c [email protected]
h ps://doi.o g/10.1016/j.de cel.2022.09.002
SUMMARY
Blood- essel o ma ion gene a es unique ascula pa e ns in each indi idual. The p inciples go e ning he
appa en s ochas ici y o his p ocess emain o be elucida ed. Using ma hema ical me hods, we ind ha he
ansi ion be ween wo undamen al ascula mo phogene ic p og ams—sp ou ing angiogenesis and ascula
emodeling—is es ablished by a shi o collec i e on - o- ea pola i y o endo helial cells in he mouse e ina.
We demons a e ha he compe i ion be ween biochemical (VEGFA) and mechanical (blood- low-induced
shea s ess) cues con ols his collec i e pola i y shi . Shea s ess inc eases ension a ocal adhesions o e -
iding VEGFA-d i en collec i e pola iza ion, which elies on ension a adhe ens junc ions. We p opose ha
ascula mo phogene ic cues compe e o egula e indi idual cell pola i y and mig a ion h ough ension shi s
ha ansla es in o issue-le el eme gen beha io s, ul ima ely leading o uniquely o ganized ascula pa e ns.
INTRODUCTION
The blood ascula ne wo k is a b anched sys em i iga ing all o -
gans in e eb a es, which is undamen al o emb yogenesis,
physiology, and healing. Dys unc ion o his ne wo k is associ-
a ed wi h mul iple diseases, including cance p og ession, dia-
be ic e inopa hies, and a e io enous mal o ma ions (Po en e
and M€
akinen, 2017). Majo axial essels a e s e eo ypical and
a e o med h ough asculogenesis (Po en e and M€
akinen,
2017). Ye , expansion o his ea ly emb yonic ne wo k h ough
angiogenesis (Po en e and M€
akinen, 2017), seems s ochas ic
because i gene a es ascula u es wi h unique pa e ns, which
can be used o biome ic iden i ica ion (Ha ung e al., 2012).
Angiogenesis in ol es wo dis inc mo phogene ic p ocesses:
(1) Sp ou ing angiogenesis, which elies on chemoa ac an s,
such as ascula endo helial g ow h ac o A (VEGFA)|: his p o-
cess expands p e-exis ing ne wo ks h ough p oli e a ion,
mig a ion, and anas omosis o endo helial cells (ECs), and o ms
imma u e ne wo ks; and (2) ascula emodeling, which elies on
blood- low-induced shea s ess|: his p ocess con e s
imma u e ne wo ks gene a ed by sp ou ing angiogenesis in o
hie a chical ascula ne wo ks, equi ing essel p uning, a e io-
enous di e en ia ion, and essel specializa ion (Ko n and
Augus in, 2015;Po en e and M€
akinen, 2017). How ECs shi
be ween hese wo mo phogene ic p ocesses, and which p inci-
ples go e n he o ma ion o well-o ganized, ye unique, ne -
wo ks emain ou s anding ques ions in ascula biology.
Sp ou ing and emodeling mo phogene ic p og ams in ol e
dynamic coo dina ion o cell pola i y and mig a ion (Fonseca
e al., 2020;Ko n and Augus in, 2015), which a e egula ed by
De elopmen al Cell 57, 2321–2333, Oc obe 10, 2022 ª2022 The Au ho (s). Published by Else ie Inc. 2321
This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
ll
OPEN ACCESS
cell-le el and issue-le el in eg a ion o chemical and mechani-
cal signals. Fo ins ance, sp ou ing angiogenesis in ol es adhe-
ens junc ion (AJ)-media ed mechano ansduc ion egula ing
collec i e mig a ion downs eam o VEGFA s imuli (Cao e al.,
2017;Ca alho e al., 2019;F iedl and Mayo , 2017;Haye
e al., 2016). In addi ion, ECs sense and espond o blood-
low-induced shea s ess by aligning, pola izing, and mig a ing
agains he low di ec ion, a phenomenon known as low-mig a-
ion coupling (F anco e al., 2015,2016;Kwon e al., 2016;Pa k
e al., 2021;Tanaka e al., 2021;Tzima e al., 2001). Despi e he
well-known c oss alk be ween VEGFA and shea s ess o egu-
la e EC sp ou ing capaci y (Chouina d-Pelle ie e al., 2013;
Gha a i e al., 2015;Song and Munn, 2011), he e is a lack o un-
de s anding o how hese inpu s a e in eg a ed a he cell le el o
shape he ascula ne wo k a he issue scale in i o. He e, we
in es iga ed in he mouse e ina how chemical (VEGFA) and me-
chanical (shea -s ess) cues in e ac o p omo e changes in
cellula beha io s unde lying he ansi ion be ween sp ou ing-
o- emodeling mo phogene ic p og ams, which we e med as
he S>R ansi ion.
RESULTS
Endo helial pola i y pa e ns de ine he S>R
ansi ion zone
To unde s and how chemical (VEGFA) and mechanical (shea -
s ess) cues es ablish he S>R ansi ion, we analyzed he
AC
D
B
Figu e 1. Vessel mo phome ics de ine he
mo phological S>R ansi ion
(A) Schema ic o he sp ou ing on - o-op ic ne e
(SF>ON) segmen a ion axis o he e inal ascula-
u e and i s ela ionship o VEGFA and shea -s ess
g adien s. Segmen a ion bins 100 and 200 (wid h
100 mm) a e depic ed as g een ec angles. V, ein;
A, a e y.
(B) Se o pa ame e s quan i ied in each bin, e med
as essel mo phome ics.
(C) P incipal componen analysis (PCA) o essel
mo phome ics in wild- ype ascula ne wo ks ol-
lowed by k-means clus e ing. Each do ep esen s
one bin in one e ina, colo coded o he co e-
sponding bin numbe . k-means clus e ing iden i ies
2 classes o objec s, class 1 (sp ou ing) and class 2
( emodeling). n = 14 e inas.
(D) Dis ibu ion o he numbe o class 1 o class 2
bins along he SF>ON axis. G ay ec angle de ines
he mo phological S>R ansi ion zone.
mouse- e ina ascula u e, whe e VEGFA
and shea s ess a e in opposi e g adien s
(Figu e 1A). To iden i y he S>R ansi ion,
we i s measu ed 7 essel mo phome ic
ea u es (Figu e S1A) in 100-mm-wide
bins along he e ina (Figu e 1A), ollowing
he sp ou ing on (SF)- o-op ic ne e
(ON) axis (Figu e 1B). P incipal componen
analysis (PCA) classi ied each bin based
on pheno ypic simila i y (see me hod de-
ails). Each bin poin was assigned o one
o he wo expec ed biological classes (sp ou ing o emodeling),
using he k-means clus e ing algo i hm (Figu e 1C). The e-
quency dis ibu ion o bins om class 1 (sp ou ing) and class 2
( emodeling) along he SF- o-ON axis de e mined a ansi ion
a 300–400 mm om he SF (Figu e 1D), co esponding o a shi
in he p edominance o class 1 o class 2 bins. Thus, his unbi-
ased quan i a i e me hod, based on mo phome ic ea u es,
cap u ed a ansi ion in ascula o ganiza ion, he mo phological
S>R ansi ion zone.
Nex , we in es iga ed how his mo phological ansi ion could
be explained a he cellula le el, based on chemical and mechan-
ical cues. Sp ou ing angiogenesis and ascula emodeling ely on
EC mig a ion (Fonseca e al., 2020;Ko n and Augus in, 2015;Po-
en e and M€
akinen, 2017). EC pola i y is a eadou o cell mig a-
ion, and bo h VEGFA and blood low induce EC pola iza ion
and mig a ion in zeb a ish and mouse (Ca alho e al., 2019;
F ancoe al.,2015;Kwone al.,2016).The e o e,wehypo hesized
ha changes in EC pola i y could unde lie he mo phological S>R
ansi ion. To in es iga e his in e ac ion, we analyzed he angle
be ween he nucleus- o-Golgi pola i y axis and he SF edge (K-
angle), o he simula ed blood- low di ec ion (F-angle), o each
EC (Figu es 2A, S2A, and S2B). The analysis was pe o med using
PolNe (Be nabeu e al., 2018) and applying me hods p e iously
es ablished in he lab (Ca alho e al., 2019;F anco e al., 2015,
2016), on he same bins (100 mm wide) used o essel mo pho-
me ics (Figu e 1A). A he SF (bin 100 mm), he K-angle cen e ed
a 90( owa d he VEGFA g adien ) wi h a na ow sca e ing
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2322 De elopmen al Cell 57, 2321–2333, Oc obe 10, 2022
(±14.8), while he F-angle had a highe dispe sion (mean 197.3±
31.7), indica ing a poo in luence o low (Figu e 2B). A 400 mm
away om he SF, blood low gained a s ong in luence on EC
pola i y, wi h an F-angle cen e ed a 180wi h a e y na ow
dispe sion compa ed wi h bin 100 mm(178.8±4.2
;p=
0.0012, Le ene es o unequal a iances). This was associa ed
wi h a signi ican shi o he mean K-angle om 90(100 mm)
o 270(400 mm) (p = 0.0016, K uskal-Wallis ANOVA es ), ep e-
sen ing an in e sion o pola i y om ‘‘ owa d’’ o ‘‘agains ’’ he SF
(Figu es 2BandS2C). F om K- and F-angles, we calcula ed K- and
F-indexes, s anding o chemoa ac an -dependen o low-
dependen pola i y indexes (PIs), espec i ely (Figu es 2Cand
S2D). K- and F-indexes quan i y he obus ness o EC pola i y o-
wa d (posi i e alues) o agains (nega i e alues) a gi en pola i y
cue (de ails in STAR Me hods). No ably, K- and F-indexes a e
an ico ela ed (R
2
=0.838), highligh ing an in e dependency
be ween he wo pola i y cues (Figu e S2E). Close o he SF, he
absolu e alue o he K-index was highe han F-index, demon-
s a ing he dominance o he chemoa ac an s imulus (0.22 ±
0.03 e sus 0.09 ± 0.07 a 100 mm, espec i ely) (Figu es 2C
and S2D). Ye , away om he SF egion, K- and F-indexes in-
e ed, wi h K-indexes becoming nega i e and lowe han
F-indexes (0.21 ± 0.04 e sus 0.32 ±0.05 a 400 mm, espec-
i ely), demons a ing he dominance o low in de e mining EC
pola i y (Figu e 2D). Thus, based on he ma hema ical desc ip ion
o EC pola i y, we de ined he cellula S>R ansi ion as a 100-mm-
wide egion (bin wid h) cen e ed a he poin whe e he mean
F-index in e sec s he mean o K-index (Figu e 2D). The global
analysis loca es he cellula S>R ansi ion a 230 ± 50 mm om
he SF (Figu e 2D), while analysis pe e ina se s i a 203.3 ±
23.0 mm(Figu e 2E). Ana omical mapping places he cellula
S>R ansi ion jus ahead o he a e y ip in he mouse e ina (Fig-
u e2F).O e all,wep oposea sys em-le elma hema icaldesc ip-
ion o EC pola i y o p edic he S>R ansi ion; ha he cellula
S>R ansi ion zone (180–280 mm) (Figu e 2D) p ecedes he
mo phological S>R ansi ion zone (300–400 mm) (Figu e 1D);
and ha he pola i y landscape o ECs is domina ed by he e ec
o shea s ess, e ealing a na ow egion o in luence o chemo-
a ac an signaling.
Compe i ion be ween VEGFA and shea s ess le els
de ines he S>R ansi ion zone
Ou analysis o he S>R ansi ion p edic s ha inc easing le els
o VEGFA o educing shea s ess should p omo e sp ou ing
Figu e 2. Collec i e EC pola i y pa e ns es ablish he cellula S>R ansi ion
(A) Rep esen a ion o he calcula ion me hod o chemoa ac an -induced (K-) and low-induced (F-) angles based on indi idual EC pola i y (nucleus- o-Golgi
ec o ) axis.
(B) Dis ibu ion o K-angles (blue) and F-angles ( ed) along he SF>ON axis. Solid line ep esen s mean, and ligh a ea ep esen s SEM.
(C) Calcula ion o K- and F-indexes, based on K- o F-angles, espec i ely.
(D) Dis ibu ion o K- (blue) and F-indexes ( ed) wi hin 500 mm om he SF (dashed box in Figu e S1C). Solid line ep esen s mean, and ligh a ea ep esen s SEM.
Dashed black line ep esen s andom pola i y. G ay ec angle de ines he cellula S>R ansi ion zone.
(E) Box plo o he cellula S>R ansi ion in each e ina. Whiske s: min. o max.
(F) Mo phological anno a ion o he cellula S>R ansi ion in a mouse e ina. Red essel co esponds o he ip o he e inal a e y. n = 11 e inas.
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De elopmen al Cell 57, 2321–2333, Oc obe 10, 2022 2323
angiogenesis. Con e sely, educ ion o VEGFA le els o
inc easing shea -s ess le els should p omo e ascula emod-
eling. In e es ingly, VEGFA and low ha e been shown o in e -
sec in in i o s udies (Vion e al., 2020). Thus, we manipula ed
blood low o VEGFA o dis u b he S>R ansi ion. Fi s , we
ea ed animals wi h cap op il (an angio ensin-con e ing
enzyme inhibi o ) o educe blood low o angio ensin-II o in-
c ease blood low (Nehme e al., 2019). Cap op il ea men led
o a signi ican inc ease in essel densi y and in he numbe o
endo helial ip cells, associa ed wi h sp ou ing angiogenesis
(Figu es 3A and S3A–S3I) and co ela ed wi h a spa ial shi on
he cellula S>R ansi ion om 230 o 325 mm(Figu e 3B).
Angio ensin-II ea men dec eased ascula densi y and he
numbe o ip cells, abolishing he S>R ansi ion zone
(Figu es 3C, 3D, and S3A–S3I). The appa en lack o ansi ion
is likely due o he sensi i i y o ou me hod, gi en he limi a ions
o bin size equi ed o s a is ical analysis. The e o e, he
absence o a clea S>R ansi ion zone should be in e p e ed
as i he ansi ion happens in he i s bin. Nex , we manipula ed
VEGFA le els in he mouse e ina by in aocula injec ion o ei he
VEGFA o sFLT1 (a VEGFA inhibi o ) (Simons e al., 2016). Simila
o cap op il, ising VEGFA le els led o an inc ease in essel
densi y and he numbe o ip cells (Figu es 3E and S3J–S3R)
and shi ed he S>R ansi ion om 230 o 260 mm(Figu e 3F).
VEGFA blockage wi h sFLT1 educed ascula densi y,
dec eased numbe o ip cells, and abolished he S>R ansi ion
(Figu es 3G, 3H, and S3J–S3R), simila o angio ensin-II, ye wi h
a s onge e ec . These e ec s we e conco dan wi h a end o
inc eased exp ession o low-sensi i e genes, Kl 2 and Kl 4, in
angio ensin-II ea men o a dec ease in cap op il ea men
(Figu e S4A). These e ec s also co ela ed wi h he dis ance
om he SF whe e Kl 4 p o ein can be de ec ed in ECs, demon-
s a ing ha angio ensin-II and cap op il ea men s lead o
inc eased o dec eased shea -s ess le els, espec i ely
(Figu es S4B and S4C). The inc eases and dec eases in shea -
s ess le els we e also e ealed by an o e all inc eased o
dec eased PI in capilla y ECs, espec i ely (Figu e S3H).
Rema kably, d ug ea men s did no a ec endogenous
VEGFA-exp ession le els, sugges ing ha ea men s do no
induce signi ican al e a ions in issue-oxygena ion le els
(Figu e S4A).
Analysis o indi idual e inas con i ms s a is ically signi ican
shi s in he S>R ansi ion zone in all condi ions, when compa ed
wi h con ol e inas (Figu e 3I). Vessel mo phome ics analyses
(independen o cell pola i y) con i med ha cap op il and
VEGFA led o an en ichmen in class 1 (sp ou ing) bins, whe eas
angio ensin-II o sFLT1 p omo ed class 2 ( emodeling) bins,
when compa ed wi h con ol e inas (Figu es 3J, 3K, and S4D).
Al oge he , hese esul s demons a e ha he cellula S>R an-
si ion is es ablished by an in e ac ion be ween blood- low
(shea -s ess) and chemoa ac an (VEGFA) g adien s, which
compe e o de ine EC collec i e beha io .
Rema kably, excessi e VEGFA le els uel abe an angiogen-
esis in pa hological condi ions, whe e essels o m imma u e
and poo ly unc ional ne wo ks (Fonseca e al., 2020). To es i
his compe i i e beha io be ween blood low and chemoa ac-
an s could also egula e pa hological angiogenesis, we used he
oxygen-induced e inopa hy (OIR) model as a means o p omo e
abe an angiogenesis (Sco and F u ige , 2010). In his assay,
mice a e exposed o hype oxic condi ions ollowed by no moxic
condi ions. Hype oxia-induced ascula eg ession leads o
pa hological neoangiogenesis pos hypoxia (Sco and F u ige ,
2010), a p ocess dependen on high VEGFA le els (Hollande s
e al., 2015). In hese condi ions, we injec ed angio ensin-II o
cap op il om day 4 pos hype oxia o 3 consecu i e days and
analyzed ascula iza ion o he e ina a day 7 pos hype oxia.
Cap op il ea men enhanced e ascula iza ion o he a ascula
a ea while angio ensin-II led o a delay in ascula iza ion
(Figu es 4A and 4B). This sugges s ha manipula ion o low-
mig a ion coupling can in luence he ou come o pa hological
angiogenesis as i does du ing de elopmen .
Mechano ansduc ion a ocal adhesions go e ns
shea -s ess-induced pola i y
Nume ous epo s p oposed ha ECs pola ize ups eam
o downs eam in esponse o low ia di e en mechanisms
(McCue e al., 2006;Tanaka e al., 2021;Tzima e al., 2003), bu
he mechanism egula ing low-mig a ion coupling emains un-
clea . Se e al shea -s ess senso s ha e been iden i ied in ECs,
including piezo1, plexinD1, ocal adhesions (FAs), he VEGF e-
cep o 2 (VEGFR2)/pla ele and endo helial cell adhesion
molecule 1 (PECAM1)/VE-cadhe in (VEcad) complex, and ca eo-
lae (Tanaka e al., 2021;Tzima e al., 2005;Xan his e al., 2019). To
cla i y how shea s ess p omo es EC pola i y, we es ablish in i o
low condi ions in which ECs pola ize agains he low di ec ion,
measu ed by he PI (Ca alho e al., 2019), in a o ce-dependen
manne (Figu es S5A and S5B), as seen in i o.Weused2.0Pa
o 4 h as ou s anda d o induce obus collec i e pola iza ion
o EC monolaye s. Flow-dependen EC pola iza ion was associ-
a ed wi h signi ican changes in FAs, as p e iously epo ed (Jalali
e al., 2001;Li e al., 1997). Exposu e o shea s ess inc eased he
numbe o FAs pe cell, inc eased hei mean leng h (Figu es 5A,
5B, S5C, and S5D), inc eased he co-localiza ion o inculin wi h
in eg in alpha 5 (ITGA5) (Figu es S5E–S5G) and o phospho yla ed
paxillin wi h inculin (Figu es S5H and S5I), and inc eased he
phospho yla ion o paxillin, inculin, ocal adhesion kinase (FAK),
and p o ein kinase B (PKB/AKT) (Figu es S6A and S6B). Rema k-
ably, shea s ess induced a bias in he alignmen o bo h FA
mo phology and he dis ibu ion o inculin-ac i a ed in eg in
be a 1 (aITGB1) co-localiza ion in he di ec ion o low
(Figu es 5C and 5D). Nex , we es ed he in ol emen o FAs in
EC pola i y esponse. Fi s , we knocked down (KD) ei he
ITGB1, ITGA5, o alin 1, as a means o dec ease FAs. Howe e ,
when applying low, KD cells eadily de ached, and measu e-
men s we e no possible. To ci cum en his echnical issue, we
used RGDS, a pep ide ha binds o in eg in RGD-binding mo i s
and blocks in eg in-media ed adhesion (Kapp e al., 2017).
RGDS- ea men impai ed FA o ma ion (Figu es S6D and S6E)
and led o a signi ican dec ease in EC pola iza ion agains he
low di ec ion (Figu e 5E). Cilengi ide, an RGDS-mimic (Kapp
e al., 2017), led o a simila e ec (Figu e 5E). Thus, FAs a e
necessa y o low-induced EC pola i y.
FAs egula e ansla ion-dependen and ansla ion-indepen-
den cellula esponses. Gi en ha inhibi ion o ansc ip ion
using ip olide, o ansla ion using pu omycin, did no
a ec low-media ed pola iza ion (Figu es S6F and S6G) hus
pola i y esponse is ansla ion-independen . FAs a e mechano -
esponsi e, sensing he igidi y, molecula composi ion, and
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2324 De elopmen al Cell 57, 2321–2333, Oc obe 10, 2022

ABCD
EFGH
IJK
Figu e 3. VEGFA and low pa e n go e n he S>R ansi ion
(A, C, E, and G) Top: scheme o designa ed compound injec ion and collec ion o samples; Bo om: ep esen a i e image o he ascula ne wo k ollowing
designa ed compound ea men .
(B, D, F, and H) Top: p edic ed s eng h o each mo phogen based on designa ed compound ea men . Bo om: dis ibu ion o K- (blue) and F- ( ed) indexes
wi hin 500 mm o he SF. Solid line ep esen s mean, and ligh a ea ep esen s SEM. Dashed g ay line ep esen s andom pola i y. G ay ec angle de ines each
compound- ea ed S>R ansi ion. Dashed g ay bounding ec angle shows con ol S>R ansi ion (de ined in Figu e 2D).
(I) Box plo o he cellula S>R ansi ion in each e ina o con ol, cap op il, angio ensin-II, VEGFA and sFLT1 e inas. n = 11 con ol; n = 9 cap op il; n = 4
angio ensin II; n = 7 VEGFA; n = 4 sFLT1. p alues om Mann-Whi ney es be ween con ol and he co esponding g oup. Whiske s: min. o max.
(J) k-means clus e ing analysis o essel mo phome ics o designa ed condi ions p ojec ed in o he bina y PCA clus e ing space de ined in Figu e 1C. Each do
ep esen s one bin o each e ina.
(K) Ra io o class 1 bins o e o al bins o designa ed condi ions. n = 14 con ol; n = 9 cap op il; n = 8 angio ensin II; n = 7 VEGFA; n = 9 sFLT1. p alues o a wo-
ailed Mann-Whi ney es be ween con ol and he co esponding g oup. E o ba s: SEM.
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De elopmen al Cell 57, 2321–2333, Oc obe 10, 2022 2325
con o ma ion o he ex acellula ma ix (ECM) (Kechagia e al.,
2019). To es i mechano ansduc ion egula es shea -s ess-
induced pola i y esponses, we manipula ed subs a e s i ness.
The ange o young modulus o ascula basemen memb anes
in i o ha e been p oposed o be in he ange o 1 kPa–1 MPa (Le-
clech e al., 2020;Wood e al., 2010). Flow-s imula ed human
umbilical ein ECs (HUVECs) seeded on 3 o 18 kPa so polydime-
hylsiloxane (PDMS) had bigge FAs han in s a ic condi ions (Fig-
u e S7A). In e es ingly, shea s ess-s imula ed ECs a 3 kPa
showed a endency o ewe FAs and a lowe PI compa ed wi h
hose a 18 kPa (Figu es S7A and S7B). The ela ionship be ween
s i ness and low-induced PI was e en mo e e iden in s i e sub-
s a es. S i e ha d PDMS (1 MPa) had highe PIs compa ed wi h
so e ha d PDMS (280 kPa), a phenomenon ha was posi i ely
associa ed wi h he leng h o FAs in he di e en condi ions
(Figu es S7C and S7D). These esul s sugges ha he abili y o
Figu e 4. E ec s o cap op il and angio ensin-II in pa hological angiogenesis
(A) Rep esen a i e images o OIR-exposed mouse e inas a 7 days a e e u n o no moxia ea ed wi h PBS, cap op il o , angio ensin-II in days 4, 5, and 6.
Re inas we e s ained o CD31 (g ay) and a ascula a eas a e highligh ed in g een.
(B) Quan i ica ion o ac ion o a ascula a ea o e o al a ea o designa ed ea men s. PBS (n = 5), cap op il (n = 6), and angio ensin-II (n = 4) mouse e inas.
p alues om one-way ANOVA wi h Tukey mul iple-compa isons es s. Whiske s: min. o max.
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2326 De elopmen al Cell 57, 2321–2333, Oc obe 10, 2022
ECs o pola ize agains he low di ec ion is dependen on he
s eng h o adhesion o he ECM. To con i m his hypo hesis, we
measu edFA-media ed ension h ough ac ion o ce mic oscopy
(TFM) (Bu le e al., 2002). As expec ed, di ec applica ion o low
on he subs a e wi hou cells does no gene a e measu able ac-
ions (Figu es S7E and S7F). TFM e ealed ha low-s imula ed
ECs exe ed signi ican ly highe le els o ac ion o ces on he
subs a e, when compa ed wi h s a ic condi ions (Figu es 5Fand
5G). Howe e , he amoun o ension pe inculin molecule, es i-
ma ed by FRET e iciency, was simila unde bo h low and s a ic
condi ions (Figu es S7G and S7H). FA ma u a ion and ac ion
o ces a e media ed by myosin-II-dependen con ac ili y (Ch za-
nowska-Wodnicka and Bu idge, 1996). Inhibi ion o bo h Rho-
associa ed p o ein kinase (ROCK) (Y-27632) and myosin ATPase
ac i i y (blebbis a in [BBS]) led o a signi ican dec ease in he
numbe and size o FAs (Figu es 5H, S7I, and S7J), and in he
co-localiza ion o inculin wi h ITGA5 (Figu e S7K), which was
associa ed wi h a dec ease in ac ion o ces, when compa ed
wi h con ol condi ions (compa e Figu es 5F and 5G wi h
Figu es S7L and S7M). These e ec s co ela ed wi h a signi ican
impai men in low-induced EC pola iza ion unde ac omyosin in-
hibi ion (Figu e 5I). Thus, ou da a sugges ha shea s ess s imu-
la es FA assembly and enhances ac ion o ces, which a e
equi ed o low-induced EC pola iza ion.
A shi in ension dis ibu ion egula es he compe i ion
be ween chemoa ac an s and shea s ess o
es ablish EC pola i y pa e ns
P e ious epo s demons a ed ha collec i e EC pola i y d i en
by chemoa ac an s in ol es ension a AJs (Ca alho e al.,
2019;Haye e al., 2016;Hu enee s e al., 2012), while ou da a
e ealed ha low-media ed pola i y is induced h ough ension
a FAs (Figu e 6A). Thus, we hypo hesize ha blood low and
chemoa ac an s migh compe e o EC pola i y by shi ing he
A
EFG
BC D
HI
Figu e 5. FA-media ed ac ion o ces d i e low-induced pola i y
(A) Rep esen a i e image o AJs (VE-cadhe in [VEcad], blue) and FAs ( inculin [g een] and ITGA5 ( ed) in s a ic o low-s imula ed HUVEC monolaye s (high
magni ica ion om Figu e S5C).
(B) Box plo o he numbe o FAs in s a ic o low condi ions. n = 8 (s a ic) and n = 6 ( low). p alue om Mann-Whi ney es . Whiske s: min. o max.
(C) Rep esen a i e supe - esolu ion image o co-localiza ion be ween inculin (g een) and ac i a ed in eg in be a 1 (aITGB1) ( ed) in s a ic o low-s imula ed
HUVEC monolaye s. Scale ba s: 2 mm.
(D) Top: angula his og ams o he dis ibu ion o inculin FA o ien a ion in s a ic and low condi ions. Bo om: mean angle o o ien a ion o he ec o om inculin
o aITGB1 cen oids in ela ion o igh - o-le slide axis in s a ic and low condi ions. Flow di ec ion is igh o le . n = 20 images om 4 sepa a e expe imen s.
(E) Box plo o pola i y index in s a ic o low-s imula ed HUVEC monolaye s ea ed wi h PBS (n = 3), RGDS (n = 5), and cilengi ide (n = 3), p alues om one-way
ANOVA wi h Sidak es . Whiske s: min. o max.
(F and G) Mean ac ion maps (F) and box plo o mean ac ion o ces (G) exe ed by s a ic o low-s imula ed HUVEC monolaye s. n = 4 s a ic; n = 5 low. p alues
om Mann-Whi ney es . Whiske s: min. o max.
(H) Box plo o numbe s o ocal adhesions in low-s imula ed HUVEC monolaye s ea ed wi h DMSO (n = 9), Y-27632 (n = 5), o BBS (blebbis a in, n = 5). p alues
om Mann-Whi ney es . Whiske s: min. o max.
(I) Box plo o pola i y index in s a ic o low-s imula ed HUVEC monolaye s ea ed wi h DMSO (n = 4), Y-27632 (n = 5) o BBS (blebbis a in, n = 6). p alues om
one-way ANOVA wi h Sidak es . Whiske s: min. o max.
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De elopmen al Cell 57, 2321–2333, Oc obe 10, 2022 2327
AB
CDE
FGHI
Figu e 6. Chemoa ac an s and blood low compe e o es ablish he main pola i y axis o ECs
(A) Schema ic o p oposed model o chemoa ac an - and low-induced collec i e pola i y o ECs.
(B) Layou o he low-chemoa ac an compe i ion in i o assay depic ing he ela ionship be ween each wound side and he low di ec ion. EC pola i y angle (a)
is measu ed in ela ion o he igh - o-le (RL) axis o he slide, in he same di ec ion o low.
(C–E) PI o designa ed egions in s a ic (blue) o low ( ed) in con ol (C), RGDS (D) and siCTNNA1 (E) HUVECs. n = 4 con ol; n = 5 RGDS; n = 7 siCTNNA1. p alues
o mul iple compa isons wi h nonin e ac i e egion one-way ANOVA wi h Sidak es . E o ba s: SD.
(F) PI o designa ed egions in s a ic o low condi ions in si-con ol ( ed) o siCTNNA1 HUVECs ea ed wi h PBS (g ay) o RGDS (blue). n = 4 pe condi ion; ed
p alues (siCTNNA1) and blue p alues (siCTNNA1 + RGDS) o mul iple compa isons wi h si-con ol one-way ANOVA wi h Sidak es . E o ba s: SD.
(G) PI o designa ed egions in s a ic (blue) o low ( ed) condi ions in HUVECs ea ed wi h DMSO o Y-27632. n = 5 pe condi ion. p alues co espond o mul iple
compa isons wi h nonin e ac i e egion one-way ANOVA wi h Sidak es . E o ba s: SD.
(H) Box plo o pe cen age o inculin colocalizing wi h in ITGA5 in low-s imula ed DMSO- o Y-27632- ea ed HUVEC monolaye s. n = 8 pe condi ion. p alues
om Mann-Whi ney es . Whiske s: min. o max.
(I) Box plo o pe cen age o inculin colocalizing wi h VE-cadhe in (VEcad) in low-s imula ed HUVEC monolaye s ea ed wi h DMSO (n = 11) o Y-27632 (n = 6).
p alues om Mann-Whi ney es . Whiske s: min. o max.
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2328 De elopmen al Cell 57, 2321–2333, Oc obe 10, 2022
Con inued
REAGENT o RESOURCE SOURCE IDENTIFIER
T ip olide Sigma-Ald ich Ca #T3652
Powe SYBR G een PCR Mas e Mix Applied Biosys ems Ca #4368702
T i on X-100 Sigma-Ald ich Ca #T8787
Vec ashield moun ing medium Vec o Labo a o ies Ca #H-1000
T ypLEExp ess Enzyme (1X) Al agene Ca #12605028
Gela in solu ion Sigma-Ald ich Ca #G1393
Penicillin/S ep omycin Gibco Ca #15140122
Phospha ase and p o einase inhibi o s
cock ail
The mo Scien i ic Ca #10085973
4x Laemmli Sample Bu e Bio- ad Ca #161-0747
DTT Sigma-Ald ich Ca #D0632
Sul o-SANPAH The mo Scien i ic Ca #22589
APTS Sigma-Ald ich Ca #A3648
FluoSphe esCa boxyla e-Modi ied
Mic osphe es beads
In i ogen Ca #F8810
Na
2
BO
4
O
7
Sigma-Ald ich Ca #221732
H
3
BO
3
Sigma-Ald ich Ca #B1934
C i ical comme cial assays
RNeasy Mic o Ki Qiagen Ca #50974004
GeneJe RNA Pu i ica ion Ki The mo Scien i ic Ca #K0702
Duolink In Si u Red Mouse/Rabbi
S a e Ki
Sigma-Ald ich Ca #DUO92101-1KT
BCA p o ein assay ki The mo Scien i ic Ca #23227
ECLWes e n Blo ing De ec ion
Reagen 24
GE Heal hca e Ca #RPN2209
Supe sc ip IV Fi s -S and Syn hesis
Sys em
In i ogen Ca #18091050
Dow Co ning 184 Sil. Elas ome Ki Ellswo h Adhesi es Ibe
´ ica SL Ca #0002-04-000002
Expe imen al models: Cell lines
HUVEC Passage 1-5 Lonza Ca #C2519A
HEK293T Passage 20-25 ATCC Ca #CRL3216
Expe imen al models: O ganisms/s ains
Mouse: Myh9 loxed Le
´on e al., 2007 N/A
Mouse: C nna1 loxed Vasioukhin e al., 2001 N/A
Mouse: Pdg biC eERT2 Clax on e al., 2008 N/A
Mouse: Cdh5C eERT2 So
¨ ensen e al., 2009 N/A
Mouse: C57BL/6 Ins i u o de Mecicina
Molecula (bo n in house)
N/A
Oligonucleo ides
Human siRNAs agains CTNNA1 Ho izon Disco e y Ca #J-010505-06
Human siRNAs agains CHD5 Ho izon Disco e y Ca #J-003641-07
Human siRNAs agains KDR Ho izon Disco e y Ca #J-003148-10
Human siRNAs agains PECAM1 Ho izon Disco e y Ca #J-017029-08
See Table S1 o addi ional in o ma ion on
oligonucleo ides used o qPCR
N/A N/A
Recombinan DNA
pRRL-VinculinTS Addgene Plasmid #111830
DR8.2 Addgene Plasmid #12263
VSVG Addgene Plasmid #45494
(Con inued on nex page)
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De elopmen al Cell 57, 2321–2333.e1–e9, Oc obe 10, 2022 e2

RESOURCE AVAILABILITY
Lead con ac
Fu he in o ma ion and eques s o esou ces and eagen s should be di ec ed o and will be ul illed by he lead con ac , Cla
´udio A.
F anco ([email p o ec ed]).
Ma e ials a ailabili y
This s udy did no gene a e new unique eagen s.
Da a and code a ailabili y
dAll da a epo ed in his pape , including mic oscopy da a o MSM da a equi ed o eanalyze he da a epo ed in his pape will
be sha ed by he lead con ac upon eques .
dO iginal code ela ed o Plexus Mo phome ics has been deposi ed a Zenodo and is publicly a ailable as o he da e o pub-
lica ion. DOI is lis ed in he key esou ces able. Ma lab sc ip s used o analyse da a in his pape is a ailable in he supplemen al
in o ma ion ile.
dAny addi ional in o ma ion equi ed o eanalyze he da a epo ed in his pape is a ailable om he lead con ac upon eques .
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mice
In his s udy, we used he ollowing mouse s ains: Myh9 loxed (Le
´on e al., 2007); C nna1 loxed (Vasioukhin e al., 2001);
Pdg biC eERT2 (Clax on e al., 2008); Cdh5C eERT2 (So
¨ ensen e al., 2009); and WT C57BL/6. C57BL/6 pups we e used o
Con inued
REAGENT o RESOURCE SOURCE IDENTIFIER
So wa e and algo i hms
ImageJ Schneide e al., 2012 h ps://imagej.nih.go /ij/
MATLAB The Ma hWo ks, Inc h ps://ma lab.ma hwo ks.com/
PolNe Be nabeu e al., 2018 h p://l c.amu.edu.pl/polne /index.php
G aphPad P ism 7 G aphPad h ps://www.g aphpad.com/
Pho oshop Adobe h ps://www.adobe.com/p /p oduc s/
pho oshop.h ml
SciPy Vi anen e al., 2020 h ps://scipy.o g/download/
Ma plo lib Hun e , 2007 h ps://ma plo lib.o g/s able/use s/
ins alling/index.h ml
Openc B adski, 2000 h ps://openc .o g/ eleases/
pyFFTW F igo, 1999 h ps://pypi.o g/p ojec /pyFFTW/
Py hon 3 Ha is e al., 2020 h ps://www.py hon.o g/downloads/
NumPy Ha is e al., 2020 h ps://numpy.o g/ins all/
Sciki -image an de Wal e al., 2014 h ps://sciki -image.o g/docs/s able/
ins all.h ml
Cy hon Behnel e al., 2011 h ps://pypi.o g/p ojec /Cy hon/
Pandas McKinney, 2010 h ps://pandas.pyda a.o g/docs/
ge ing_s a ed/ins all.h ml
Ima is 9.8 Ox o d Ins umen s h ps://ima is.oxins .com/
Vessel Mo phome ics This s udy h ps://doi.o g/10.5281/zenodo.7036288
O he
Cy ocen icO2 Con olle BioSphe ix, USA Ca #P oOx 110
EGM-2 Bulle ki Lonza Ca #CC-3162
Leibo i z L15 media Li e echnologies, LTI Ca #21083-027
iBIDI m-Slide I 0.4 Lue iBIDI Ca #80176
Minipuls3 pe is al ic pump Gilson Ca #GM3P4
4-15% Mini-PROTEANTGX Ge Bio-Rad Ca #456-1084
DAPI Li e Technologies Ca #D1306; RRID: AB_2629482
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e3 De elopmen al Cell 57, 2321–2333.e1–e9, Oc obe 10, 2022
modula ion o low, VEGF a ailabili y o d ug ea men . No dis inc ion o sex o animals was conside ed in his s udy. Mice we e
main ained and b eed a he Ins i u o de Medicina Molecula Joa
˜o Lobo An unes (IMM-JLA) unde s anda d husband y condi ions
and unde Po uguese egula ions. Fo ins ance, mice we e housed ou o i e pe cage, and allowed ee access o wa e and
ood. B eedings we e pe o med in duos o ios, and pups we e kep all ime wi h mo he s du ing ea men s, wi h he excep ion
o OIR expe imen s, whe e pups we e kep only he mo he .
E hics s a emen
Animal expe imen a ion was ca ied ou in compliance wi h EU Di ec i e 86/609/EEC and Recommenda ion 2007/526/EC ega ding
he p o ec ion o animals used o expe imen al and o he scien i ic pu poses. Animal p ocedu es we e pe o med unde supe ision
by he IMM-JLA Animal E hics Commi ee (ORBEA), unde he p ojec licenses AWB_2015_10_CF_Pola idade and AWB_2021_02_
CF_Vascula , and app o ed by he Po uguese Animal E hics Commi ee egula o y body (DGAV), p ojec licenses 0421/000/000/
2016 and 0421/000/000/2021.
Cul u e o HUVECs
Human umbilical ein endo helial cells – HUVECs (Lonza, C2519A) – we e cul u ed ollowing he manu ac u e ’s guidelines, in il e -
cap T75 lasks Nunclon Dsu ace ea men (VWR in e na ional, LLC) wi h comple e medium EGM-2 Bulle ki (Lonza, CC-3162) a
37C and 5% CO
2
o ensu e s able en i onmen o op imal cell g ow h. All he expe imen s we e conduc ed wi h HUVECs be ween
passages 1 and 5. When passaging HUVECs o expe imen s, cells we e washed wice in s e ile PBS (137mM NaCl, 2.7mM KCl,
4.3mM Na
2
HPO
4
, 1.47mM KH
2
PO
4
, pH7.4) and hen incuba ed o 5min in T ypLEExp ess Enzyme (1X) (Al agene, 12605028)
a 37C, 5% CO
2
. When 95% o he cells de ached, comple e medium was added o each lask o inhibi he ac i i y o he
T ypLEExp ess Enzyme and he cell suspension was ans e ed in o a alcon ube. Cells we e hen cen i uged a 700 pm o
5min a RT and he pelle e-suspended in esh comple e medium. HUVECs we e hen seeded a he desi ed concen a ion, depend-
ing on he expe imen s.
METHOD DETAILS
In i o mouse ea men s
Fo inc easing blood low, angio ensin II (Sigma-Ald ich, A9525) was injec ed in ape i oneally (IP) 10mL/g (10 mg/mL s ock solu ion)
daily a pos na al day 3 (P3), P4 and P5 and pups we e collec ed a P6. On he opposi e, o educe blood low, cap op il (Sigma-
Ald ich, C4042) was injec ed IP 15mL/g (3.3mg/mL s ock solu ion) daily a P3, P4 and P5 and pups we e collec ed a P6. In bo h ex-
pe imen s, con ol mice we e injec ed using PBS alone. Fo in aocula adminis a ion o eagen s, P4-P5 pups we e anes he ized
and in a i eal injec ions we e pe o med unde a s e eomic oscope using a 10ml Hamil on sy inge equipped wi h a 33-gauge needle.
App oxima ely 0.5ml o s e ile solu ion o PBS was injec ed pe eye, while he con ala e al eye emained uninjec ed. The ollowing
subs ances we e used: ecombinan VEGFA (493-VE-050; R&D Sys ems, 3mg/ml) o ecombinan VEGFR1/Fl -1 Fc chime a p o ein
(sFLT1; 471-F1-100; R&D Sys ems, 1mg/ml). Mice we e sac i iced 36h la e . The eyes we e emo ed, ixed in 2% pa a o maldehyde
(PFA, Sigma-Ald ich, 4412244) in PBS a 4C o 5h, insed in PBS and p ocessed o IHC. In he case o Y-27632 ea men , Y-27632
(Me ck Millipo e, 688001) was injec ed IP (10mg/kg) a P5 and pups we e sac i iced a P6.
In gene dele ion expe imen s, 4-hyd oxy amoxi en (Sigma-Ald ich, H6278) was injec ed IP (20 mg/g) a P1 and P3 o Myh9 loxed
mice and a P4 o C nna1 loxed, and eyes we e collec ed a P6. As con ols, C e-nega i e li e ma es we e used in all expe imen s.
Bo h males and emales we e used, wi hou dis inc ion.
In OIR expe imen s, P8 pups and hei nu sing mo he s we e housed in o an ai - igh chambe and exposed o 75% oxygen le el in
ai main ained by a P oOx 110 oxygen con olle (BioSphe ix, USA). They we e e u ned o oom ai a P11, also named D0 o day
0 a e e u n o no moxia. In OIR animals, Cap op il (C4042, Sigma Ald ich, Ge many) (50 mg/g), Angio ensin II (A9525, Sigma Ald ich,
Ge many) (100 mg/g) o ehicle (PBS) was injec ed IP a D4, D5, and D6 and pups we e sac i iced a D7. Eyes we e collec ed and ixed
wi h 2% PFA in PBS o e nigh a 4C.
Vi al p oduc ion and ansduc ion
Replica ion-incompe en len i i uses we e p oduced by ansien ans ec ion o HEK293T wi h len i i al exp ession ec o co- ans-
ec ed wi h he i al packaging ec o DR8.2 and he i al en elope ec o VSVG. Medium was eplaced wi h esh cul u e medium
6-8h pos ans ec ion. 48h a e medium eplacemen , len i i al pa icles we e concen a ed om supe na an by ul acen i uga ion
a 112.500g o 1h30 and e-suspended in 0.1% BSA PBS. Seeded HUVECs we e ansduced wi h a ying concen a ions o a len-
i i al plasmid con aining pRRL-VinculinTS (Ro henbe g e al., 2018). 24h a e i al ansduc ion he cul u e medium was eplaced by
esh comple e medium and cells we e kep unde cul u e condi ions up un il 72h pos - ansduc ion and hen p ocessed o imaging.
Immuno luo escence on mouse e inas
Re inas we e s ained as p e iously desc ibed (F anco e al., 2013). B ie ly, e inas we e incuba ed o 2h, a RT in Claudio’s Blocking
Bu e (CBB). Fo pola i y expe imen s, e inas we e incuba ed wi h an i-ICAM2 (1:100, BD Biosciences 553326) and an i-ERG (1:200,
Abcam ab92513) p ima y an ibodies in 1:1 PBS and CBB mix u e a 4C o/n. On he nex day, e inas we e washed 3 imes (30min
each) wi h PBST (PBS wi h 0.1% T i on X-100, Sigma-Ald ich, T8787) and u he incuba ed wi h seconda y an ibodies an i- a Alexa
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De elopmen al Cell 57, 2321–2333.e1–e9, Oc obe 10, 2022 e4
555 (The mo Fishe Scien i ic, A21434) and an i- abbi Alexa 488 (The mo Fishe Scien i ic, A21206) in 1:1 PBS and CBB mix u e o/n
a 4C. The day a e , e inas we e washed h ee imes (30min each) wi h PBST and incuba ed wi h Fab
2
agmen s (Donkey an i- ab-
bi , Jackson ImmunoResea ch, 711-006-152) o 2h a RT wi h gen le shaking. A e wa ds e inas we e ixed wi h 4% pa a o malde-
hyde (PFA) a RT o 15min and washed h ee imes (15min each) wi h PBST, ollowed by 1h incuba ion wi h CBB a RT. Nex , e inas
we e incuba ed wi h an i-GOLPH4 p ima y an ibody (1:400, Abcam, ab28049) in 1:1 PBS and CBB mix u e a 4C o/n. The day a e ,
e inas we e washed h ee imes (30min each) wi h PBST and incuba ed wi h an i- abbi Alexa 647 (The mo Fishe Scien i ic, A31573)
seconda y an ibody in 1:1 PBS and CBB o/n a 4C. Then, e inas we e washed h ee imes (30min each) wi h PBST and la -moun ed
on glass slides using Vec ashield moun ing medium (Vec o Labo a o ies, H-1000). Addi ional an ibodies we e used: an i-CD31
(R&D, AF3628, 1:200), an i-pPaxilin-Y118 (Cell Signaling, 2541S, 1:100), an i-Cdh5 (BD biosciences, 555289, 1:25), an i-Kl 4
(R&D, AF3158, 1:50). Images we e acqui ed by ile-scans wi h mul iple Z-posi ions using a Zeiss Cell Obse e SD (Ca l Zeiss) equip-
ped wi h Zen so wa e and wi h a Plan-Apoch oma 40x NA 1.40 oil o Plan-Apoch oma 63x NA 1.40 objec i es.
siRNA ans ec ion
In o de o silence he exp ession o genes o in e es , a se o ON-TARGET human siRNAs agains CTNNA1 (Dha macon, GE Heal h-
ca e, J-010505-06), CDH5 (Dha macon, GE Heal hca e, J-003641-07) o un a ge ing con ol we e used. B ie ly, HUVECs we e
seeded he day be o e he ans ec ion o each 60-70% con luence and we e hen ans ec ed wi h 25nM o siRNA using he
Dha maFECT 1 eagen (Dha macon, GE Heal hca e) ollowing he Dha macon siRNA T ans ec ion P o ocol. 24h a e ans ec ion
he cul u e medium was eplaced by esh comple e medium and cells we e kep unde cul u e condi ions up un il 72h pos - ans-
ec ion and hen p ocessed o u he expe imen s.
Flow mic o luidic assays
Fo he low mic o luidics assay, HUVECs we e pla ed a a concen a ion o 3x10
6
cells/mL on o iBIDI m-Slide I
0.4
Lue (iBIDI, 80176),
p e iously coa ed wi h 0.2% gela in solu ion in H
2
O (Sigma-Ald ich, G1393), 4h p io low applica ion. When ans ec ed cells we e
used, hey we e pla ed 68h a e ans ec ion. Flow cul u e medium consis ed o Leibo i z L15 media (Li e echnologies, LTI 21083-
027) supplemen ed wi h EGM-2 SingleQuo s
TM
(Lonza, CC-4176) and 1% penicillin/s ep omycin (Gibco, #15140122). A e 4h o
a achmen , HUVECs we e subjec ed o low a di e en anges, depending on he expe imen , o 4 hou s o he app op ia e amoun
o ime. The iBIDI slides we e connec ed o a pe is al ic pump (Gilson Minipuls3) ha ensu es a con inuous lamina low du ing he
expe imen s.
Fo he low and sc a ch wound-assay se o expe imen s, HUVECs we e pla ed on o mic oscopy glass slides (The mo Scien i ic,
76x26mm) coa ed wi h 0.2% gela in solu ion in H
2
O (Sigma-Ald ich, G1393) a 3x10
6
cells/mL. When HUVECs eached con luence, a
wound was c ea ed by sc a ching he su ace o he mic oscopy glass slide wi h a 200mL pipe e ip. The cul u e medium was hen
eplaced by esh comple e medium and HUVECs we e allowed o mig a e unde low, using s icky-Slide I
0.4
Lue (iBIDI, 80178) o
5 hou s.
When assessing he e ec s o VEGFA and KDR in pola i y assays, Leibo i z L15 media supplemen ed wi h 50ng/mL hVEGFA (P e-
po ech) was used ins ead o EGM-2 SingleQuo s.
D ugs assays
Fo he expe imen s wi h inhibi o s, HUVECs we e seeded a 3x10
6
cells/mL in iBIDI m-Slide I
0.4
Lue (iBIDI, 80176), p e iously coa ed
wi h 0.2% gela in solu ion in H
2
O (Sigma-Ald ich, G1393), 4h p io low applica ion. Inhibi o s we e added o he media 1h be o e he
low mic o luidic assay a speci ic concen a ions. When indica ed, cells we e ea ed wi h ()-Blebbis a in (20mM, Sigma-Ald ich,
B0560), Y-27632 (5mM, Me ck Millipo e, 688001), RGDS (20mM, Toc is, 3498), Pu omycin (200mg/ml, Sigma-Ald ich, P8333) and
T ip olide (2mM, Sigma-Ald ich, T3652).
Immuno luo escence o cul u ed HUVECs
Fo immuno luo escence o in i o cul u ed HUVECs, cells we e ixed in 1% PFA *Sigma-Ald ich, 4412244) in PBS o 30min a RT.
Fixed HUVECs we e blocked and pe meabilized wi h blocking solu ion con aining 3% BSA, 0.1% T i on X100 in PBS o 30min a RT.
Then cells we e incuba ed o 2h a RT wi h he app op ia e p ima y an ibodies dilu ed in he blocking solu ion (an i-VE-cadhe in, R&D
- AF938, 1:50; an i-GOLPH4, Abcam - ab28049, 1:400; an i-Vinculin, Sigma-Ald ich - V9264, 1:400; an i-ITGA5, Abcam - ab150361,
1:100; an i-pPaxillin, Cell Signaling - 2541S, 1:100; an i-ac i a ed ITGB1 BD Pha mingen - 553715, 1:100) and washed 3 x 15min wi h
PBST. A e wa ds, cells we e incuba ed in blocking solu ion con aining he seconda y luo opho e conjuga ed an ibodies o 1h a RT
in he da k (Donkey an i-goa Alexa 647, The mo Fishe Scien i ic - A21447, 1:400; Donkey an i-mouse Alexa 488, The mo Fishe
Scien i ic - A21202, 1:400; Donkey an i- abbi Alexa 568, The mo Fishe Scien i ic - A10042, 1:400), ollowed again by 3 washes
o 15min in PBST. Finally, HUVECs we e incuba ed wi h 1x DAPI (Molecula P obes by Li e Technologies, D1306) dilu ed in PBS
o 5min in he da k. Fo mo phological o colocaliza ion analysis, high- esolu ion Z-s ack images a mul iple posi ions we e acqui ed
on a con ocal Lase Poin -Scanning Mic oscope 880 (Zeiss) equipped wi h he Zen black so wa e wi h a Plan Apoch oma 63x NA
1.40 oil DIC M27 objec i e. Fo pola iza ion analysis, images a mul iple posi ions we e acqui ed on Zeiss Axio e 200M equipped
wi h an EC Plan-NeoFlua 40x NA 0.75.
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P o ein ex ac ion and wes e n blo ing
P o ein ex ac ion was pe o med om HUVECs lysed in 120mL o RIPA bu e supplemen ed wi h phospha ase and p o einase in-
hibi o s cock ail (1:100, Fische Scien i ic, #10085973). Adhe en cells we e hen de ached om he pla e wi h a cell sc appe and he
cell lysa es we e ans e ed in o an ice cold eppendo ube and cen i uged a maximum speed o 10min a 4C. P o ein concen-
a ion was quan i ied using he BCA p o ein assay ki (Pie ce
TM
, The mo Fishe Scien i ic, 23227) ollowing he manu ac u e ’s guide-
lines. The Mul imode mic opla e eade , In ini e M200 (TECAN), was used o spec opho ome ic measu emen o p o ein wi h he
i-con olso wa e. Fo Wes e n Blo ing, p o ein samples we e no malized up o 25mL and combined wi h a mix u e o 4x Laemmli
Sample Bu e (Bio- ad Labo a o ies, #161-0747) wi h 450mM DTT (SigmaAld ich, D0632) and incuba ed a 95C o 5min. P o ein
samples we e loaded and sepa a ed on a 4-15% Mini-PROTEANTGX Gel (BioRad, #456-1084) along wi h 5mL o p o ein ladde (GE
Heal hca e, RPN800E). A e ans e , blo ed memb anes we e incuba ed in Ponceau Red o assess ans e quali y, and hen
washed in TBST (50mM T is/HCl, 150mM NaCl, 0.1% Tween-20, pH7.5). A e wa ds, memb anes we e incuba ed in blocking solu-
ion con aining 3% BSA in TBST o 1h a RT, ollowed by an o/n incuba ion a 4C wi h he p ima y an ibodies dilu ed in he same
blocking bu e , an i-pPaxillin Y118 (Cell Signalling - 2541S, 1:1000), an i-ßac in (Sigma, A5441, 1:5000), an i-Vinculin (Sigma-Ald ich,
V9264, 1:1000), an i-pVinculin Y100 (The mo Fishe Scien i ic, 44-1074G, 1:1000), an i-VE-cadhe in (San a C uz Bio echnology, sc-
9989, 1:1000), an i-aCa enin (Sigma-Ald ich, C2081, 1:1000), an i-Talin(Cell Signaling, 4021, 1:1000), an i-AKT (Cell Signaling, 9272,
1:1000), an i-pAKT S473 (Cell Signaling, 4060, 1:1000), an i-FAK (Cell Signaling, 71433, 1:1000), an i-pFAK Y397 (Cell Signaling,
8556, 1:1000), an i-NF-kB p65 (Cell Signaling, 6956, 1:1000) and an i-pNF-kB p65 S536 (Cell Signaling, 3033, 1:1000). On he
ollowing day memb anes we e washed 3 imes in TBST and incuba ed wi h seconda y ho se adish pe oxidase (HRP)-conjuga ed
an ibodies o 1h a RT. Be o e e ela ion, memb anes we e washed again 3 imes in TBST o 5min and hen incuba ed in ECL
Wes e n Blo ing De ec ion Reagen 24 (GE Heal hca e, RPN2209) ollowing he manu ac u e ’s p o ocol. P o ein bands we e isu-
alized in Ame sham 680 (Cy i a) and ela i e p o ein quan i ies we e measu ed using FIJI so wa e (Schneide e al., 2012).
RNA ex ac ion and cDNA syn hesis
RNA ex ac ion om HUVECs. HUVECs RNA ex ac ion using he RNeasy Mic o Ki (Qiagen) and he GeneJe RNA Pu i ica ion Ki
(The mo Scien i ic) as desc ibed by he manu ac u e ’s p o ocol. RNA concen a ion was quan i ied using NanoD op 1000 (The mo
Scien i ic) and adjus ed equally, ollowed by DNase I diges ion (The mo Scien i ic) and cDNA syn hesis (Supe sc ip IV Fi s -S and
Syn hesis Sys em, In i ogen). In some cases, a spike-in RNA con ol was added o he mix u e in o de o con ol o gene exp ession
du ing he eal- ime quan i a i e PCR (RT-qPCR). cDNA samples we e hen dilu ed in RNAse/DNAse- ee wa e o he subsequen
RT-qPCR eac ions.
RNA ex ac ion om mouse e inas. Pai o e inas om he same animal we e dissec ed in ice-cold esh PBS and immedia ely
snap ozen in liquid ni ogen. Re inas we e lysed in RLT bu e om he RNeasy Mic o Ki (Qiagen) o exed igo ously and u he
homogenized using 30g sy inge needles. Lysa es we e cen i uged o clea om p ecipi a es, and supe na an was used o RNA
ex ac ion as desc ibed by he manu ac u e ’s p o ocol, including in column DNase I ea men . RNA concen a ion was quan i ied
using NanoD op 1000 (The mo Scien i ic) and adjus ed equally cDNA syn hesis (Supe sc ip IV Fi s -S and Syn hesis Sys em, In i-
ogen). cDNA samples we e hen dilu ed in RNAse/DNAse- ee wa e o he subsequen eal- ime quan i a i e PCR (RT-qPCR)
eac ions.
RT-qPCR
RT-qPCR was pe o med using a 7500 Fas Real-Time PCR Sys em (Applied Biosys ems) wi h Powe SYBR G een PCR Mas e Mix
(Applied Biosys ems) ollowing he s anda d p og am o he sys em p e iously men ioned. Fo each eac ion, 5mL o cDNA was com-
bined wi h 10mL o Powe SYBR G een PCR Mas e Mix, 4.5mL o RNAse/DNAse ee wa e and 0.5mLo 4mM p ime s pool
(Fo wa d+Re e se) in a Mic oAmp Fas Op ical 96-well Reac ion Pla e (Applied Biosys ems).
The exp ession le els o each sample duplica e we e hen no malized o GAPDH o spike-in RNA and he 2
-DDT
me hod was used o
calcula e ela i e al e a ions in gene exp ession. P ime s used in his pape a e p o ided as Table S1.
PDMS gels
PDMS (Polydime hylsiloxane) was p oduced wi h di e en s i ness. B ie ly, Silicone Elas ome (Sylga d 184 Silicone Elas ome , Dow
101697) was mixed wi h cu ing agen (Sylga d 184 Silicone Elas ome , Dow 101697) a h ee di e en a ios (by weigh ): 5:1; 10:1 and
20:1, co esponding o a Young’s elas ic modulus o 1000kPa; 580kPa and 280kPa, espec i ely, ollowed by degassing in a acuum
chambe o 30min a RT and cu ed by hea ing in an o en a 75C o 2h, as p e iously epo ed (Pa k e al., 2010). A e polyme -
iza ion, PDMS gels we e demolded and s e ilized. A e wa d, gels we e coa ed wi h 0.2% gela in solu ion in H
2
O o 30min a
37C and HUVECs seeded as desc ibed be o e o low expe imen s.
So -PDMS gels wi h di e en s i nesses we e p oduced o measu e he ocal adhesion leng h in ECs unde shea s ess. B ie ly,
solu ion A and B (DOWSILCY 52-276 A&B, Dow) we e mixed in a 1:1 o 5:6 (w/w) a io co esponding o a bulk elas ic modulus o
3kPa and 18.6 kPa espec i ely, ollowed by degassing in a acuum chambe o 30min on ice. A e wa ds, 2ml o so -PDMS we e
added o glass slides and spun o 90s, 24V in a handmade spin-coa e . Finally, slides wi h so -PDMS we e cu ed by hea ing in an
o en a 65C o/n.
To p omo e be e cell adhesion, so -PDMS gels we e incuba ed wi h 0.2mg/ml o Sul o-SANPAH (sul osuccinimidyl 6-(4’-azido-
2’-ni ophenylamino) hexanoa e, The mo Scien i ic) wice du ing 5 min unde UV lamp (app oxima ely 365nm) and washed wice wi h
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OPEN ACCESS
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50mM o HEPES (Gibco). Finally, so -PDMS gels we e coa ed wi h 0.2% gela in solu ion in H
2
O o 30min a 37C and HUVECs
seeded 4h p io low expe imen s as desc ibed p e iously.
T ac ion o ce mic oscopy and monolaye s ess mic oscopy
Fo he TFM expe imen s, so -PDMS gels (1:1 a io) we e p oduced as desc ibed p e iously (Pa k e al., 2010). To coa wi h
luo escen beads, 5% APTES (SIGMA, A3648) was used o silanise he so -PDMS and he gels we e washed h ee imes wi h
E OH absolu e. The glass slides wi h he so -PDMS we e hen d ied in an o en a 60C o 10min. FluoSphe esCa boxyla e-
Modi ied Mic osphe es beads (In i ogen, F8810) we e dilu ed (1:50) in a bo ic solu ion (Na
2
BO
4
O
7
, SIGMA 221732 and H
3
BO
3
,
SIGMA B1934), il e ed wi h a 0.45mm il e and sonica ed o 10min. A e wa ds, luo escen beads we e added o he so -PDMS
gels and incuba ed o 5min a RT. Gels we e hen d ied in an o en a 60C. Then, gels we e incuba ed wi h 0.2mg/ml o Sul o-
SANPAH [sul osuccinimidyl 6-(4’-azido-2’-ni ophenylamino) hexanoa e, The mo Scien i ic] wice du ing 5 min unde UV lamp
(app oxima ely 365nm) and washed wice wi h 50mM o HEPES (Gibco). Finally, so -PDMS gels we e coa ed wi h 0.2% gela in
solu ion in H
2
O o 30min a 37C and HUVECs seeded 4h p io low expe imen s.
T ac ion o ce measu emen s we e pe o med as desc ibed p e iously (Bu le e al., 2002). Fo each condi ion, luo escen images
o cell monolaye s and nanobeads placed on he su ace o he gels we e imaged in Leica SP8 mul i-pho on mic oscope using he
Insigh DS+ Dual pulsed lase a 920nm and using a Leica 20x objec i e (NA 0.95) using ile scans o 4x4 ields-o - iew. A he end o
he measu emen s, cells we e de ached om he gel wi h 10x ypsin/EDTA (Gibco) and an image o bead posi ion in he elaxed s a e
o he gel was acqui ed. Indi idual iles o each ield o iew we e s i ched by using FIJI (Schneide e al., 2012) and he G id/Collec ion
s i ching plugin (P eibisch e al., 2009). 2D images o he de o med subs a e we e compa ed o he elaxed s a e wi h a cus om-made
PIV so wa e in Ma lab (Ma hWo ks) o ob ain he 2D de o ma ion o he op laye o he gel. Finei e- hickness Fou ie - ans o m ac-
ion o ce mic oscopy was hen used o calcula e he ac ion o ces exe ed by he cells (Elosegui-A ola e al., 2014;T epa e al.,
2009). The a e age o ces pe uni a ea exe ed by each monolaye we e hen calcula ed. To calcula e he minimum de ec able o ce
le els, we ollowed he same p ocedu e in cell- ee gel a eas, and calcula ed he esul ing o ces.
Monolaye S ess Mic oscopy (Tambe e al., 2011,2013) was used o calcula e he monolaye ension om he ac ion ields. I
was implemen ed, as a cus om-made so wa e, in Py hon 3 using NumPy (Ha is e al., 2020), SciPy (Vi anen e al., 2020), Ma plo lib
(Hun e , 2007), sciki -image ( an de Wal e al., 2014), pandas (McKinney, 2010), pyFFTW (F igo, 1999), openc (B adski, 2000) and
cy hon (Behnel e al., 2011).
P oximi y liga ion assay
A e low mic o luidic expe imen s, HUVECs we e p ocessed o PLA using he Duolink In Si u Red Mouse/Rabbi S a e Ki (Sigma-
Ald ich, DUO92101-1KT) as desc ibed by he manu ac u e ’s p o ocol. To p obe in e ac ions be ween VINCULIN and VE-cadhe in,
cells we e incuba ed wi h an an i- inculin an ibody aised in abbi (Sigma-Ald ich, V4139) and an an i-VE-cadhe in an ibody aised in
mouse (San a C uz Bio echnologies, sc-9989). In pa allel, cells we e also incuba ed wi h an an i-VE-cadhe in an ibody aised in goa
(R&D Sys ems, AF938) and subsequen ly wi h an an i-goa Alexa 647 seconda y an ibody (The mo Fishe Scien i ic, A21447) o label
adhe ens junc ions. To p obe in e ac ions be ween VINCULIN and ITGA5, cells we e incuba ed wi h an an i- inculin an ibody aised
in mouse (Sigma-Ald ich, V9264) and an an i-ITGA5 an ibody aised in abbi (Abcam, ab150361).
To quan i y colocaliza ion o PLA signal a adhe ens junc ions, high- esolu ion Z-s ack images a mul iple posi ions we e acqui ed
on a con ocal Lase Poin -Scanning Mic oscope 880 (Zeiss) equipped wi h he Zen black so wa e wi h a Plan Apoch oma 63x NA
1.40 oil DIC M27 objec i e. B ie ly, PLA do s we e quan i ied using ImageJ’ pa icle analysis ool and he da a no malized by he num-
be o cells.
QUANTIFICATION AND STATISTICAL ANALYSIS
Tension senso FRET measu emen s
Cells in ec ed wi h he i al plasmid pRRL-VinculinTS (Ro henbe g e al., 2018) we e used o hese expe imen s. FRET images we e
ob ained using a con ocal Lase Poin -Scanning Mic oscope 880 (Zeiss) equipped wi h a Plan-Apoch oma 63x, NA 1.40, oil imme -
sion, DIC M27 objec i e and an a gon lase ea u ing 405, 458 and 514nm lase lines. Fo FRET expe imen s, an MBS 458/514 beam
spli e was used in combina ion wi h he ollowing il e s: mTFP1 GaAsP, band-pass 461–520; Venus/FRET, band-pass 525–575.
Accep o pho obleaching expe imen s we e analyzed using a cus om w i en MATLAB sc ip . A Gaussian il e wi h s anda d de i-
a ion o 0.75 was applied o he images be o e analysis. The in ensi y in he egion o in e es was measu ed be o e and a e bleach-
ing. FRET e iciency was calcula ed as EF =Ipos Ip e
Ipos , whe e Ipos and Ip e a e he in ensi y o he dono channel a e and be o e
bleaching espec i ely.
Quan i ica ion o Focal Adhesions and Colocaliza ion Analysis
To quan i y numbe and size o ocal adhesions, co-immunos ainings o inculin and ITGA5 oge he wi h VE-cadhe in we e pe -
o med o iden i y s uc u es ha sha ed inculin and ITGA5 s aining. The numbe o hese s uc u es we e hen quan i ied pe
cell, de ined by he VE-cadhe in con ou . To quan i y size o ocal adhesions, inculin-ITGA5 s uc u es we e selec ed and he inculin
s aining only was hen used o pe o m measu emen s.
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Fo colocaliza ion analyses, high- esolu ion Z-s ack con ocal images o HUVECs s ained o di e en p o eins (VE-Cadhe in,
Vinculin and ITGA5) we e impo ed and analyzed in MATLAB using a cus om w i en code (coloc_FA, p o ided in supplemen al in-
o ma ion as Da a S1). An objec -based co-localiza ion app oach was pe o med. Concisely, each channel was segmen ed, and a
bina y mask gene a ed. The masks we e combined and he ac ion o pixels wi h o e lapping signals was quan i ied.
Analysis o adhesion o ien a ion and inculin-aITGB1 colocaliza ion angle
To analyze FA o ien a ion, high esolu ion Z-s ack con ocal images o HUVECs s ained o inculin and aITGB1 we e segmen ed and
bina ized using ImageJ and hen impo ed and analyzed in MATLAB using a cus om w i en code (coloc_FA, p o ided in supple-
men al in o ma ion as Da a S1). An objec -based analysis app oach was pe o med. B ie ly, each inculin objec o ien a ion was
compu ed o de e mine FA adhesion o ien a ion. Each inculin and aITGB1 objec cen oid coo dina ed we e de e mined. Each in-
culin objec was pai ed o he closes aITGB1 objec , and hei espec i e cen oids used o de e mine a inculin- o-aITGB1 ec o .
Vec o angles we e compu ed o de e mine he pola iza ion o aITGB1 ela ed o inculin.
Vessel and endo helial densi y analysis
Fo adial expansion quan i ica ion, images we e aken using an EC Plan-Neo lua , NA 0.30, ai 10x objec i e, in a con ocal Lase
Poin -Scanning Mic oscope 880 (Zeiss). The mig a o y leng h o he ascula plexus was analyzed by measu ing he o al leng h
o he e inal ascula u e om he op ic ne e, in he cen e , owa ds he e inal pe iphe y – sp ou ing on .
Fo he quan i ica ion o he numbe o ip cells, ile-scan images o he whole sp ou ing on we e aken wi h a C-Apoch oma Co ,
NA 1.20, wa e 40x objec i e in a con ocal Lase Poin -Scanning Mic oscope 880 (Zeiss). The numbe o ip cells was hen coun ed,
and he alues no malized by he sp ou ing on leng h.
Rega ding essel densi y, ile-scan images o he whole pe al we e acqui ed using a Plan-Apoch oma , NA 0.8, ai 20x objec i e in
a con ocal Lase Poin -Scanning Mic oscope 880 (Zeiss). A e essel segmen a ion using FIJI and Pho oshop, essel densi y was
calcula ed as a a io be ween essel a ea and o al a ea o he pe al.
Endo helial cell densi y was calcula ed using 20x ile-scan images acqui ed in a con ocal Lase Poin -Scanning Mic oscope 880
(Zeiss). The numbe o ERG
+
nuclei coun ed manually was no malized by he ascula ized a ea o each pe al ( wo pe als o each
e ina).
Vascula mo phogenesis pa ame e s and p incipal componen analysis
Re inal ascula plexuses we e imaged, bina ized, and skele onized ollowing he p o ocol p e iously desc ibed in Be nabeu e al.
(2018). Vessel diame e was s o ed as a node a ibu e in he g aph da a s uc u e. Plexus Mo phome ics we e calcula ed using
a MATLAB sc ip de eloped in his s udy (h ps://doi.o g/10.5281/zenodo.7036288). In de ail, he esul ing plana g aphs we e manu-
ally c opped o he a e ial egion o in e es and simpli ied by me ging edges ha me a nodes o deg ee 2. E e y emaining e ex in
he g aph was he e o e a bi u ca ion (deg ee 3), he ip o blind-ended essel (sp ou o essel unde going p uning) (deg ee 1) o
essel lea ing he egion o in e es (deg ee 1). The aces o he plana g aph we e ob ained, and hei a ea calcula ed. The sp ou ing
on bounda y was de ined as he line connec ing he ips o he wo sp ou s p o uding he mos in he sp ou ing on . Wi h his line
as e e ence, he ollowing g aph p ope ies we e calcula ed in bins o 100 mm wid h mo ing away om he sp ou ing on : essel
densi y (numbe o g aph edges pe uni o ascula ized a ea in he bin), bi u ca ion densi y (numbe o deg ee 3 nodes pe uni o
ascula ized a ea in he bin), ange o a ascula a eas (di e ence be ween maximum and minimum a ea o he aces calcula ed in
he bin), mean a ascula a ea (mean o he dis ibu ion o ace a eas in he bin), s anda d de ia ion o a ascula a eas (s anda d de-
ia ion o he dis ibu ion o ace a eas in he bin), mean diame e (mean o he dis ibu ion o essel diame e s in he bin), and s an-
da d de ia ion o diame e s (s anda d de ia ion o he dis ibu ion o essel diame e s in he bin). P incipal componen analysis (PCA)
decomposi ion o he 7-dimensional ec o s de ining he p e ious ea u es a each bin in each con ol e ina was pe o med o acil-
i a e isualiza ion and bin classi ica ion based on pheno ypic simila i y. Weigh s we e as ollowing: PCA1 [-0.453; -0.345; 0.348;
0.366; 0.404; -0.406; -0.304]; PCA2 [0.346; 0.0638; 0.523; 0.220; 0.491; 0.423; 0.366], o essel densi y; bi u ca ion densi y; ange
o a ascula a eas; mean a ascula a ea; s anda d de ia ion o a ascula a eas; mean diame e ; and s anda d de ia ion o diame e s,
espec i ely. The k-means clus e ing algo i hm was used o ind wo clus e s ha minimize wi hin clus e a iance unde he assump-
ion ha wo main pheno ypic classes (sp ou ing and emodeling) exis . The PCA decomposi ion o he con ol g oup was used o
map he bins o he emaining g oups o he same pheno ypic space. The k-means classi ie was used o classi y hese bins acco d-
ing o hei dis ance o he cen e o he sp ou ing/ emodeling clus e s in he con ol g oup.
Pola i y index quan i ica ions in i o
To quan i y cell pola i y, ile-scan images o HUVECs s ained o Golgi (Golph4) and nuclei (DAPI) ma ke s we e p ocessed in FIJI.
A e wa ds, each se o images was impo ed and analyzed in MATLAB using a modi ied e sion o a pola i y analysis sc ip kindly
p o ided by Anne-Cle
´mence Vion and Holge Ge ha d (PI_calcula o , p o ided in supplemen al in o ma ion as Da a S2).
B ie ly, a e segmen ing each channel co esponding o he Golgi and nuclea s aining, he cen oid o each o ganelle was
de e mined and a ec o connec ing he cen e o he nucleus o he cen e o i s co esponding Golgi appa a us was d awn. The
Golgi-nucleus assignmen was done au oma ically minimizing he dis ance be ween all he possible couples. The pola i y o each
cell was de ined as he angle be ween he ec o and he slide axis. An angula his og am showing he angle dis ibu ion was
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De elopmen al Cell 57, 2321–2333.e1–e9, Oc obe 10, 2022 e8
hen gene a ed. Ci cula s a is ics we e pe o med using he Ci cula S a is ic Toolbox. The pola i y index (PI) was calcula ed as he
leng h o mean esul an ec o o a gi en angula dis ibu ion.
pola i y index =ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1
NX
N
1
cosɑ!2
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NX
N
1
sinɑ!2
u
u
The PI a ies be ween 0 and 1. The close o 1, he mo e he da a a e concen a ed a ound he mean di ec ion, while alues close o
0 co espond o andom dis ibu ion. PI indica es he collec i e o ien a ion s eng h o he cell monolaye . Box plo s we e gene a ed
by using e e y single PI calcula ed o images o each biological eplica, which show he biological a iabili y o he sys em. This da a
was used o calcula e he signi icance o di e ences be ween expe imen al condi ions.
Flow and chemoa ac an pola i y indexes in e inas
In he in i o pola i y analysis, we quan i ied pola i y and co ela ed i wi h blood low di ec ion by using he app oach desc ibed in
Be nabeu e al. (2018). B ie ly, all he e inal ascula plexuses we e imaged in a Zeiss Cell Obse e SD (Zeiss) equipped wi h Zen
so wa e and wi h a Plan-Apoch oma 40x NA 1.40 oil objec i e and pos -p ocessed o gene a e a bina y mask om he ICAM2 chan-
nel and a second image wi h a leas he ERG (EC nuclei) and Golph4 (Golgi appa a us) channels. These wo images de ined he inpu
o PolNe . PolNe is a g aphical use in e ace ha allows he use o pe o m h ee asks: 1) o cons uc a low model om he ICAM2
mask and use he HemeLB low sol e o es ima e he wall shea s ess ac oss he whole ne wo k (as well as blood eloci y, shea
a e, and p essu e); 2) o d aw cell pola i y ec o s (nuclei o Golgi) o each endo helial cell in he ne wo k based on he E g-Golph4
image; and 3) o s a is ically analyze he ela ionship be ween he cell pola i y and low di ec ion o wall shea s ess.
Chemoa ac an (K-) and Flow (F-) pola i y indexes we e hen calcula ed using he angle ha each Nuclei- o-Golgi ec o does
wi h ei he he sp ou ing on edge (K-), de ined as a line be ween he wo mos ou wa d ascula sp ou s, o he low di ec ion
(F-) using he leng h o mean esul an ec o o a gi en angula dis ibu ion, as p e iously desc ibed (Be nabeu e al., 2018;Ca alho
e al., 2019;F anco e al., 2015,2016). The K- and F-indexes we e calcula ed using 3 consecu i e Ma lab sc ip s gene a ed in he lab
(AnalysisSubRegion_Scale, Me ge_Regions and Me ge_All, all p o ided in supplemen al in o ma ion as Da a S3). (The pola i y index
a ies be ween -1 (backwa d pola iza ion – away om he sp ou ing on , i K-index; o wi h he low di ec ion, i F-index) o 1 ( o wa d
pola iza ion – owa ds ee edge o he sp ou ing on , i K-index; o agains he low di ec ion, i F-index), whe e 0 means andom
pola iza ion. Chemoa ac an (K-) and Flow (F-) pola i y indexes we e hen ep esen ed as a unc ion o he dis ance om he sp ou -
ing on (SF) o he op ic ne e (ON).
S a is ical analysis
All s a is ical analysis was pe o med using G aphPad P ism 7. Measu emen s we e aken om dis inc samples, and s a is ical de-
ails o expe imen s a e epo ed in he igu es and igu e legends. Sample size is epo ed in he igu e legends and no s a is ical es
was used o de e mine sample size. The biological eplica e is de ined as he numbe o cells, images, animals, as s a ed in he igu e
legends. No inclusion/exclusion o andomiza ion c i e ia we e used and all analyzed samples a e included. No mali y es s we e pe -
o med o assess he da a no mali y. Compa isons be ween wo expe imen al g oups we e analyzed wi h wo-sided unpai ed pa a-
me ic es o Mann-Whi ney es depending on he da a no mali y. Mul iple compa isons be ween mo e han wo expe imen al
g oups we e assessed wi h one-way ANOVA. We conside ed a esul signi ican when p<0.05. Fo all box plo s: cen e line, median; +,
mean; whiske s, min o max.
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