Fig. 1. tr ls by (a) Represen - Nature · 2017-02-16 · eks of HFD re ale bars repr o‐positive ce. Endotheli thin the adv moxifen‐indu vealed adve esent 100 µm lls. ... d at

Suppleme

FACS histo

confirmed

model (no

tamoxifen

WT and t

CD45 (AP

events de

0.09 ± 0.0

versus en

entary Fig. 1.

ogram of per

d that >50% o

ot presented

n‐induced end

tamoxifen‐ind

PC conjugated

etected in the

05 % (n = 4),

d.SclCreERT;R

. Specificity o

ripheral leuko

of all periphe

). (b) Repres

d.SclCreERT;R

duced, HFD‐f

d) and Yfp (d

e FITC channe

which did n

R26RstopYfp;A

of endothelia

ocytes from a

eral blood leu

sentative FAC

R26RstopYfp m

fed end.SclCr

detected in F

el in end.SclC

ot differ from

ApoE‐/‐).

al cell fate tr

dult Tie2Cre;

kocytes expr

CS histogram

mice. (c) FACS

reERT;R26Rsto

FITC channel

CreERT;R26Rst

m WT contro

racking mode

;R26RstopYfp

ess Yfp (seen

of periphera

S scatter plot

opYfp;ApoE‐/‐

). Of CD45+

topYfp;ApoE‐/

ol mice (0.07

els by FACS.

mice. Prelim

n in FITC chan

al blood leuk

s of peripher‐ mice at 18

cells, the me/‐ mice at 18

± 0.07; n = 3

(a) Represen

minary experim

nnel) in this m

kocytes from

ral leukocytes

weeks of ag

ean percenta

weeks of ag

3; p = 0.76 fo

tative

ments

mouse

adult

s from

ge for

age of

e was

or WT

Suppleme

fate track

sections f

(correspo

Immunofl

protocol,

18 weeks

entary Fig. 2.

king model. (

from tamoxife

nding to 14

luoresence st

performed o

of HFD. L = lu

. Sensitivity a

a ‐ c) Immun

en‐induced en

4, 24 or 36

taining for Yfp

n thoracic ao

umen; scale b

and specificit

ofluoresence

nd.SclCreERT;

weeks of a

p and CD31, a

ortic sections

bars represen

ty of end.SclC

e staining for

T;R26RstopYfp

age). L = lu

acquired usin

from a contr

t 100 µm.

CreERT;R26Rs

Yfp and CD3

p;ApoE‐/‐ mice

umen; scale

ng identical m

rol mouse tha

stopYfp;ApoE

1 performed

e after 8, 18 o

bars repres

microscope se

at received pe

E‐/‐ endothelia

on thoracic

or 30 weeks o

sent 100 µm

ttings and sta

eanut oil and

al cell

aortic

of HFD

m. (d)

aining

d after

Suppleme

results

end.SclCre

(FSC) and

panel), th

Following

CD31 PE‐C

entary Fig. 2

for the d

eERT;R26Rsto

d side scatter

hen live cells

g this selectio

Cy7 antibody

(continued).

etermination

opYfp;ApoE‐/‐

r (SSC) (uppe

by gating for

on, the lower

and the prop

(e) Represen

n of the

mice after 8

er left panel),

r Hoechst 33

r right panel

portion of CD

ntative FACS

proportion

weeks of HF

, then we se

342 positive

shows the p

31+ cells expr

plots demon

of CD31+

FD. Cell select

elected the s

and 7AAD ne

ercentage of

ressing Yfp.

strating the g+ cells exp

tion was first

single cell fra

egative cells

f CD31+ cells

gating schem

pressing Yf

based on fo

action (upper

(lower left p

labeled with

e and

p in

rward

r right

panel).

h anti‐

Suppleme

CD68, did

weeks of

entary Fig. 2

d not reveal

HFD. L = lume

2 (continued)

any co‐positi

en; scale bars

. (f) Immuno

ive cells at a

s represent 10

ofluoresence

ny time‐poin

00 µm.

staining for

nt. Shown he

Yfp and CD4

ere is staining

45, or (g) Yfp

g of mice aft

p and

ter 18

Suppleme

end.SclCr

CD68+ m

expressio

weeks of

entary Fig

reERT;R26Rsto

macrophages

n by αSma+

HFD. L = lume

g. 3. T

opYfp;ApoE‐/‐

in atherosc

cells in athe

en; scale bars

Tgf‐β exp‐ mice. (a) Im

clerotic plaqu

rosclerotic p

s represent 10

pression i

mmunofluores

ue. (b) Imm

laque. Sectio

00 µm. Arrow

in athero

scence stainin

munofluoresc

ons shown ar

ws indicate co

osclerotic

ng showing T

cence stainin

re from mice

o‐positive cell

plaques

Tgf‐β expressi

ng showing

e that receive

ls.

from

on by

Tgf‐β

ed 18

Suppleme

fibroblast

aortic sec

(c) 30 we

lumen; sc

indicate c

entary Fig. 4

t‐like cells wi

ctions from ta

eks of HFD re

cale bars repr

co‐positive ce

4. Endotheli

ithin the adv

amoxifen‐indu

evealed adve

resent 100 µm

lls.

ial lineage‐d

ventitia. (a –

uced end.SclC

entitial Yfp+ ce

m. Insets are

erived cells

c) Immunoflu

CreERT;R26Rs

ells co‐expres

shown at hi

undergo En

uorescence c

stopYfp;ApoE‐

ssing the fibr

gher magnifi

ndMT and g

confocal micr‐/‐ mice fed w

roblast‐specif

cation as ind

give rise to

oscopy of tho

with (a) 8, (b)

fic marker Fa

dicated and a

Fap+

oracic

18 or

p. L =

rrows

Suppleme

weeks of

Quantitat

aboveme

cells co‐e

system (th

± 0.8% an

taking int

respective

endotheli

evaluated

and 18 w

analyses.

entary Fig. 4

HFD 33.4 ±

tion (not acc

ntioned perio

xpressed Yfp

he % of endo

nd 7.3 ± 3.3%

to account th

ely, we deter

al lineage‐de

d from each o

weeks; n = 4

Analysis by 1

(continued).

5.7%, 37.3

ounting for t

ods of HFD 4.

p. (f) After ta

othelial cells e

% of adventit

he efficiency

rmined that

erived Fap+ ce

of at least 3 sp

mice for 30

1‐way ANOVA

(d) Quantita

± 3.8% and

the % of end

.9 ± 1.5%, 2.7

aking into acc

expressing Yfp

tial Fap+ cells

of our endot

5.1 ± 1.7%,

ells. Staining w

patially separ

0 weeks). Da

A.

ation of adve

29.3 ± 10.7%

dothelial cell

7 ± 0.3% and

count the eff

p), at the sam

s were derive

thelial lineage

3.2 ± 0.4% a

was performe

rated thoracic

ta were ave

ntitial cells re

% (respective

ls expressing

2.2 ± 1.0% (r

ficiency of ou

me time‐point

ed from endo

e tracking sy

and 2.2 ± 1.1

ed at each tim

c aortic sectio

eraged per an

evealed that

ely) of cells e

g Yfp) identif

respectively)

ur endothelia

ts respectivel

othelial linea

ystem, at the

1% of all adv

me‐point with

ons per mous

nimal then u

after 8, 18 a

expressed Fa

fied that afte

of adventitia

al lineage tra

y, 15.9 ± 5.0%

age cells. (g)

same time‐p

ventitial cells

h at least 4 im

se (n = 5 mice

used for stat

nd 30

p. (e)

er the

l Fap+

acking

%, 8.8

Again

points

were

mages

e for 8

tistical

Suppleme

fibroblast

of thoraci

8, (b) 18

intimal pl

indicated

entary Fig. 5

t‐like cells wi

ic aortic secti

or (c) 30 we

aques. L = lu

and arrows i

5. Endothelia

ithin the intim

ions from tam

eeks of HFD

umen; scale b

ndicate co‐po

al lineage‐de

ma and adve

moxifen‐indu

revealed Yfp+

bars represen

ositive cells.

erived cells

entitia. (a – c

ced end.SclCr+ cells co‐exp

nt 100 µm. In

undergo End

c) Immunofluo

CreERT;R26Rst

pressing the

nsets are sho

dMT and gi

orescence co

topYfp;ApoE‐/

fibroblast ma

own at highe

ve rise to F

onfocal micro/‐ mice fed wi

arker Fsp‐1 w

er magnificati

Fsp‐1+

scopy

ith (a)

within

ion as

Suppleme

and 30 w

indicative

cells expr

1.9 ± 0.6

efficiency

same tim

were der

endotheli

1.2%, 4.5

was perfo

separated

Data were

entary Fig. 5

eeks of HFD

e of a fibrobla

essing Yfp) id

6% (respectiv

y of our endo

e‐points, we

rived from e

al lineage tra

± 1.5% and 3

ormed at eac

d thoracic aor

e averaged pe

(continued).

18.4 ± 1.8%,

ast phenotyp

dentified that

vely) of intim

othelial lineag

determined

endothelial li

acking system

3.0 ± 0.9% of

ch time‐point

rtic sections p

er animal the

(d) Quantita

24.7 ± 5.0%

e. (e) Quanti

after the abo

mal Fsp‐1+ ce

ge tracking sy

that 19.1 ± 6

ineage cells.

m, at the sam

all intimal ce

t with at leas

per mouse (n

en used for sta

ation of cells

and 47.9 ± 4

tation (not a

ovementione

lls co‐expres

ystem (the %

6.7%, 18.4 ±

(g) Again t

me time‐poin

lls were endo

st 4 images e

n = 5 mice for

atistical analy

in intimal pla

4.9% (respect

ccounting fo

ed periods of

ssed Yfp. (f)

% of endothel

6.1% and 6.3

taking into a

nts respective

othelial lineag

evaluated fro

r 8 and 18 we

yses. Analysis

aques reveale

tively) of cell

r the proport

HFD 5.9 ± 2.0

After taking

ial cells expr

3 ± 1.8% of i

account the

ely, we dete

ge‐derived Fs

om each of a

eeks; n = 4 m

s by 1‐way AN

ed that after

s expressed F

tion of endot

0%, 5.6 ± 1.9%

into accoun

essing Yfp), a

ntimal Fsp‐1+

efficiency o

rmined that

sp‐1+ cells. Sta

at least 3 spa

mice for 30 we

NOVA. **P < 0

8, 18

Fsp‐1,

thelial

% and

nt the

at the + cells

of our

3.5 ±

aining

atially

eeks).

0.01.

Suppleme

from thor

8, (i) 18 o

lumen; sc

indicate c

entary Fig. 5

racic aortic se

or (j) 30 wee

cale bars repr

co‐positive ce

(continued).

ections of tam

eks of HFD al

resent 100 µm

lls.

. (h – j) Imm

moxifen‐indu

so revealed Y

m. Insets are

munofluoresce

ced end.SclCr

Yfp+ cells co‐

shown at hi

ence confoca

reERT;R26Rst

‐expressing th

gher magnifi

al microscopy

topYfp;ApoE‐/‐

he fibroblast

cation as ind

y of the adve/‐ mice fed wi

marker Fsp‐

dicated and a

entitia

th (h)

1. L =

rrows

Suppleme

fibroblast

of thoraci

8, (b) 18 o

intimal pl

Suppleme

positive c

entary Fig. 6

t‐like cells wi

ic aortic secti

or (c) 30 wee

aques. L = lum

entary Fig. 6c

ells.

6. Endothelia

ithin the intim

ions from tam

eks of HFD re

men; scale ba

c. Insets are

l lineage‐der

ma and adve

moxifen‐indu

vealed Yfp+ c

ars represent

shown at hig

rived cells un

entitia. (a – c

ced end.SclCr

cells co‐expre

100 µm for S

gher magnific

ndergo EndM

c) Immunofluo

CreERT;R26Rst

essing the fibr

Supplementar

cation as ind

MT and give

orescence co

topYfp;ApoE‐/

roblast marke

ry Figs. 6a an

icated and a

rise to Vime

onfocal micro/‐ mice fed wi

er Vimentin w

d b, and 50 µ

rrows indicat

entin+

scopy

ith (a)

within

µm for

te co‐

Suppleme

and 30 w

Vimentin,

endotheli

22.3 ± 7.9

into acco

expressin

10.4% of

account t

we deter

lineage‐de

evaluated

all time‐p

ANOVA. *

entary Fig. 6

weeks of HFD

, indicative o

al cells expre

9% and 10.1

ount the effi

g Yfp), at the

intimal Vim

he efficiency

mined that 2

erived Vimen

d from each o

points). Data

**P < 0.01.

(continued).

D 34.9 ± 6.0

f a fibroblast

essing Yfp) id

± 3.2% (resp

ciency of ou

e same time

entin+ cells

of our endot

25.8 ± 2.5%,

ntin+ cells. St

of at least 3 s

were averag

(d) Quantita

0%, 24.7 ± 3

t phenotype.

entified that

pectively) of i

ur endothelia

‐points, we d

were derived

thelial lineag

13.9 ± 2.8%

taining was

spatially sepa

ged per anim

ation of cells

.9% and 32.

(e) Quantita

after the ab

ntimal Vimen

al lineage tra

determined t

d from endo

e tracking sy

% and 10.6 ±

performed a

rated thorac

mal then used

in intimal pla

2 ± 4.1% (re

ation (not ac

ovementione

ntin+ cells co‐

acking system

that 73.8 ± 7

othelial lineag

ystem, at the

± 3.4% of all

at each time

ic aortic sect

d for statistic

aques reveale

espectively) o

counting for

ed periods of

‐expressed Y

m (the % of

7.3%, 56.5 ±

ge cells. (g)

same time‐p

intimal cells

e‐point with

ions per mou

cal analyses.

ed that after

of cells expr

the proporti

f HFD 23.8 ±

fp. (f) After t

f endothelial

11.2% and 3

Again taking

points respect

s were endot

at least 4 im

use (n = 5 mi

Analysis by 1

8, 18

ressed

ion of

3.3%,

taking

l cells

32.9 ±

g into

tively,

thelial

mages

ce for

1‐way

Suppleme

from thor

8, (i) 18 o

lumen; sc

indicate c

entary Fig. 6

racic aortic se

or (j) 30 week

cale bars repr

co‐positive ce

(continued).

ections of tam

ks of HFD also

resent 100 µm

lls.

. Immunofluo

moxifen‐indu

o revealed Yfp

m. Insets are

orescence co

ced end.SclCr

p+ cells co‐exp

shown at hi

onfocal micro

reERT;R26Rst

pressing the

gher magnifi

oscopy of the

topYfp;ApoE‐/‐

fibroblast ma

cation as ind

e adventitia (/‐ mice fed wi

arker Vimenti

dicated and a

h – j)

th (h)

in. L =

rrows

Suppleme

Sm22α+ o

microscop

fed with (

αSma+, Sm

entary Fig. 7

or Smmhc+ c

py of thoracic

(a) 8, (b) 18

m22α+ or Smm

7. Endothelia

cells in mur

c aortic secti

or (c) 30 wee

mhc+ cells in t

al lineage‐de

ine atherosc

ons from tam

eks of HFD re

this murine m

rived cells g

clerotic lesio

moxifen‐induc

evealed that

model. L = lum

give rise to a

ons. (a – c)

ced end.SclCr

Yfp+ cells giv

men; scale bar

a small prop

Immunofluo

reERT;R26Rsto

ve rise to a s

rs represent 1

portion of α

rescence con

opYfp;ApoE‐/‐

mall proporti

100 µm.

Sma+,

nfocal ‐ mice

ion of

Suppleme

and 30 w

Quantitat

the above

αSma+ ce

tracking s

1.3 ± 1.4%

(g) Again

points res

were end

images ev

mice for a

way ANOV

entary Fig. 7

eeks of HFD

tion (not acco

ementioned

ells co‐expres

system (the %

%, 1.3 ± 0.9%

taking into ac

spectively, w

othelial linea

valuated from

all time‐point

VA.

(continued).

6.1 ± 3.8%, 6

ounting for th

periods of H

ssed Yfp. (f)

% of endothel

and 7.8 ± 3.7

ccount the ef

e determined

ge‐derived α

m each of at

s). Data were

(d) Quantita

6.8 ± 0.8% an

he proportion

FD 0.4 ± 0.4

After taking

ial cells expre

7% of intimal

fficiency of ou

d that 0.08 ±

αSma+ cells. St

least 3 spatia

e averaged pe

ation of cells

nd 9.9 ± 1.7%

n of endothel

4%, 0.4 ± 0.3

into accoun

essing Yfp), at

αSma+ cells w

ur endothelia

± 0.09%, 0.09

taining was p

ally separated

er animal the

in intimal pla

% (respectivel

lial cells expre

3% and 2.4 ±

t the efficien

t the same tim

were derived

al lineage trac

9 ± 0.06% and

performed at

d thoracic ao

n used for sta

aques reveale

y) of cells ex

essing Yfp) id

± 1.1% (respe

ncy of our e

me‐points, w

d from endoth

cking system,

d 0.8 ± 0.4%

each time‐po

ortic sections

atistical analy

ed that after

xpressed αSm

dentified that

ectively) of in

endothelial lin

we determined

helial lineage

at the same

of all intima

oint with at le

per mouse (

yses. Analysis

8, 18

ma. (e)

t after

ntimal

neage

d that

e cells.

time‐

l cells

east 4

(n = 5

s by 1‐

Suppleme

and 30 we

(i) Quanti

after the

Sm22α+ c

tracking s

2.0 ± 1.3%

cells. (k) A

time‐poin

were end

4 images

mice for a

way ANOV

entary Fig. 7

eeks of HFD

itation (not a

abovementio

cells co‐expre

system (the %

%, 2.9 ± 0.7%

Again taking i

nts respective

othelial linea

evaluated fro

all time‐point

VA.

(continued).

10.7 ± 5.3%,

accounting fo

oned periods

essed Yfp. (j)

% of endothel

% and 2.5 ± 0

into account

ely, we determ

age‐derived S

om each of at

s). Data were

(h) Quantita

12.5 ± 0.8% a

or the propor

of HFD 0.6 ±

After taking

ial cells expre

0.8% of intim

the efficiency

mined that 0

m22α+ cells.

t least 3 spat

e averaged pe

ation of cells

and 15.2 ± 3.

rtion of endo

0.4%, 0.9 ± 0

g into accoun

essing Yfp), at

mal Sm22α+ c

y of our endo

.2 ± 0.1%, 0.4

Staining was

tially separate

er animal the

in intimal pla

4% (respectiv

othelial cells

0.2% and 0.8

nt the efficie

t the same tim

cells were de

othelial lineag

4 ± 0.08% an

performed a

ed thoracic a

n used for sta

aques reveale

vely) of cells

expressing Y

± 0.2% (respe

ncy of our e

me‐points, w

erived from e

ge tracking sy

nd 0.4 ± 0.1%

at each time‐

ortic sections

atistical analy

ed that after

expressed Sm

Yfp) identified

ectively) of in

endothelial lin

we determined

endothelial lin

ystem, at the

% of all intima

point with at

s per mouse

yses. Analysis

8, 18

m22α.

d that

ntimal

neage

d that

neage

same

l cells

t least

(n = 5

s by 1‐

Suppleme

vessels. C

3b, fed a c

3a and 3b

scale bars

entary Fig. 8

Control end.Sc

chow diet, we

b. No apprecia

s represent 10

8. Lack of ox

clCreERT;R26R

ere stained w

able signal wa

00 µm.

xidative stre

RstopYfp;Apo

with the ident

as seen for ei

ss and cell a

oE‐/‐ mice of t

tical DHE (a) a

ither DHE or T

apoptosis in

he same age

and TUNEL (b

TUNEL staini

non‐athero

as those sho

b) staining pro

ng in these sa

sclerotic (co

own in Figs. 3

otocols as pe

amples. L = lu

ntrol)

3a and

r Figs.

umen;

Suppleme

induce En

while H2O

per condi

induction

n = 3 per

presented

0.0001.

entary Fig. 9.

ndMT, TGF‐β d

O2 caused a d

tion. (b) Cell

was unchang

condition. Da

d in Supplem

Cellular effe

did not affect

ose‐responsi

proliferation

ged by TGF‐β

ata in Supple

mentary Table

ects of TGF‐β

t the total nu

ve reduction

assessed by

β, while H2O2

mentary Fig.

e 1. ns not sig

and H2O2 on

mber of cells

in cell count

BrdU incorpo

caused a dos

9 were analy

gnificant, *P

HUVECs. (a)

s in culture (p

t during EndM

oration durin

se‐responsive

yzed by 2‐way

< 0.05, **P

At the descri

per well of 6‐w

MT induction

g the initial 2

e reduction in

y ANOVA wit

< 0.01, ***P

ibed doses us

well culture p

over 5 days.

24 hours of E

n cell prolifer

h complete r

P < 0.001, **

sed to

plate),

n = 3

ndMT

ation.

esults

**P <

Suppleme

assessed

indicated

expected

weight/siz

by immun

EndMT in

H2O2 only

entary Fig. 9

by Western b

concentratio

molecular w

ze is 37kDa. (

nostaining for

nduction) follo

y, or TGF‐β plu

(continued).

blot for activa

ons. Activate

eight/size is

(d) Endotheli

r active Caspa

owing treatm

us 200µM H2O

(c) Endotheli

ated Caspase

ed Caspase

17kDa. GAPD

al cell apopto

ase 3 (after 4

ment with end

O2. Scale bars

al cell apopto

3 protein exp

3 ladder (L)

DH ladder rep

osis in respon

8 hours and 5

dothelial grow

s represent 10

osis in respon

pression in H

) represents

presents 37kD

nse to EndMT

5 days EndMT

wth media a

00 µm.

nse to 6 hours

UVECs with H

20kDa. Act

Da. GAPDH ex

T induction in

T induction) a

lone (Ctrl), T

s EndMT indu

H2O2 applied a

tivated Caspa

xpected mole

n HUVECs ass

and TUNEL (5

TGF‐β only, 20

uction

at the

ase 3

ecular

sessed

5 days

00µM

Suppleme

induction

complete

0.001, **

TGF‐β and

resulting m

at earlier

entary Fig. 9

. n = 4 per

results prese

**P < 0.0001

d/or H2O2 for

mesenchyma

time‐points a

(continued).

condition. D

ented in Supp

1. Note that w

5 days, to ev

al cells), exper

as indicated.

(e) H2O2, but

ata in Supple

plementary T

while in other

valuate the ef

riments b, c, e

not TGF‐β, in

ementary Fig

Table 1. ns no

r EndMT expe

ffects of these

e, and some o

ncreased cell

g. 9 were an

ot significant,

eriments end

e agents on t

of d in Supple

death after 4

nalyzed by 2‐

, *P < 0.05, *

dothelial cells

the EndMT pr

ementary Fig.

48 hours of E

‐way ANOVA

**P < 0.01, *

were treated

rocess (rather

9 were perfo

ndMT

A with

**P <

d with

r than

ormed

Suppleme

HUVECs. (

increased

involved i

in Supple

Suppleme

for qRT‐PC

entary Fig. 1

(a, b) Relative

by both TG

nduction of E

ementary Fig

entary Table 4

CR.

0. Oxidative

e RNA expres

GF‐β and H2O

EndMT by cel

g. 10 were

4. ns not sign

stress augm

ssion of Calpo

O2 in an addi

l stimulation

analyzed by

nificant, *P <

ments EndMT

onin and Vers

tive fashion.

with TGF‐β a

y 2‐way ANO

0.05, **P < 0

T and induces

ican (respect

All experim

and/or H2O2 o

OVA with co

0.01, ***P < 0

s TGF‐β path

tively) assesse

ents in Supp

over 5 days (s

omplete res

0.001, ****P

hway activati

ed by qRT‐PC

plementary F

ee methods)

ults present

< 0.0001. n =

ion in

R was

ig. 10

. Data

ed in

= 6 ‐ 9

Suppleme

increased

TGF‐β, wh

SMAD3 R

Suppleme

days (see

results pr

****P < 0

entary Fig. 1

by both TGF

hile H2O2 had

RNA expressi

entary Fig. 10

e methods). D

resented in S

0.0001. n = 6 ‐

10 (continue

F‐β and H2O2

d a marginal

on was incr

0 involved ind

Data in Supp

Supplementar

‐ 9 for qRT‐PC

ed). (c) Relat

2 in an additi

effect. (e) SM

eased by H2

duction of En

plementary F

ry Table 4. n

CR.

tive SNAI2 R

ve fashion. (

MAD2 expres

2O2, but was

ndMT by cell

ig. 10 were

ns not signific

RNA expressi

d) SNAI1 RNA

sion was not

s decreased

stimulation w

analyzed by

cant, *P < 0.

on assessed

A expression

t affected by

by TGF‐β. A

with TGF‐β a

2‐way ANOV

.05, **P < 0.

by qRT‐PCR

was increas

TGF‐β or H2O

All experimen

nd/or H2O2 o

VA with com

.01, ***P < 0

R was

ed by

O2. (f)

nts in

over 5

mplete

0.001,

Suppleme

pSMAD3,

Western b

Quantitat

both TGF

expected

molecular

weight/siz

stimulatio

analyzed

*P < 0.05,

entary Fig. 1

SMAD3 and

blots for SMA

tion (below ri

F‐β and H2O2

molecular w

r weight/size

ze is 37kDa.

on with TGF‐

by 2‐way ANO

, **P < 0.01, *

10 (continued

GAPDH on ex

AD3 protein l

ight) of West

2 causing act

weight/size i

e is 52kDa.

All experime

β and/or H2O

OVA with com

***P < 0.001,

d). (g) Weste

xposure of HU

evels, showin

tern blots sho

ivation of SM

is 48kDa. SM

GAPDH lad

ents in Supp

O2 over 5 da

mplete result

, ****P < 0.00

ern blots (ab

UVECs to TGF

ng that H2O2

owing pSMA

MAD3. pSMA

MAD3 ladder

dder represe

lementary Fi

ys (see meth

s presented i

001. n = 3 ‐ 6

bove) showin

F‐β and/or H2

but not TGF‐

D3 protein le

AD3 ladder (

r (L) represe

ents 37kDa.

g. 10 involve

hods). Data i

in Supplemen

for Western

ng protein ex

O2. Quantitat

‐β increases S

evels (relative

L) represents

ents 50kDa.

GAPDH ex

ed induction

n Supplemen

ntary Table 4.

blots.

xpression lev

tion (below le

SMAD3 in HU

e to SMAD3),

s 50kDa. pSM

SMAD3 exp

pected mole

of EndMT b

ntary Fig. 10

. ns not signif

els of

eft) of

UVECs.

, with

MAD3

pected

ecular

by cell

were

ficant,

Suppleme

HUVECs.

indicated

pathways

the right o

entary Fig. 1

(a) Pathway

that at the

s in HUVECs. S

of the vertica

1. Oxidative

y analysis pe

doses used

Significant up

l red line.

stress cause

erformed on

for EndMT

pregulation is

es inflammat

n upregulate

induction TG

indicated if t

tory and fibr

d genes ide

GF‐β alone di

the horizonta

rotic pathwa

entified from

id not upreg

al (blue) pathw

ay upregulati

m microarray

gulate any sp

way bar cros

ion in

data

pecific

ses to

Suppleme

number o

The 10 m

the horizo

entary Fig. 11

of pathways,

most significan

ontal (blue) p

1 (continued

with many o

ntly upregula

athway bar c

). (b) In cont

of these bein

ated pathway

rosses to the

trast, TGF‐β+

g related to

ys are presen

right of the v

H2O2 caused

oxidative str

nted. Significa

vertical red li

a significant

ess, inflamm

ant upregulat

ne.

upregulation

ation and fib

tion is indica

n of a

brosis.

ated if

Suppleme

HCAECs. (

TGF‐β plu

and a pro

expressio

represent

entary Fig. 1

(a) Immunos

us 200µM H2O

ogressive incre

n (in this ca

t 100 µm.

2. Oxidative

taining of HC

O2, indicating

ease in EndM

ase, SM22α)

stress incre

CAECs treated

induction of

MT with loss o

with TGF‐β,

ases EndMT

d with endot

cellular stres

of Ve‐Cadheri

and particu

and is addi

thelial growth

s with positiv

n staining an

ularly TGF‐β

tive to the e

h media alon

ve staining fo

d gain of me

plus 200µM

effect of TGF

ne (Ctrl), TGF

r active Casp

senchymal pr

H2O2. Scale

F‐β in

F‐β, or

ase 3,

rotein

e bars

Suppleme

Versican (

(g, h) Rela

decreased

by both T

was incre

Suppleme

days (see

results pr

****P < 0

entary Fig. 12

(respectively)

ative RNA exp

d by TGF‐β an

GF‐β and H2O

ased by both

entary Fig. 12

e methods). D

resented in S

0.0001. n = 6 i

2 (continued)

) in HCAECs a

pression of CD

nd H2O2. (i) R

O2 in an addit

h TGF‐β and H

2 involved ind

Data in Supp

Supplementar

in all experim

). (b ‐ f) Relat

assessed by q

D31 and TIE2

Relative RNA

ive fashion. (j

H2O2, but unli

duction of En

plementary F

ry Table 5. n

ments.

tive RNA exp

qRT‐PCR in re

(respectively

expression of

j) Relative RN

ike SNAI2 this

ndMT by cell

ig. 12 were

ns not signific

ression of FA

esponse to tr

y) assessed by

f SNAI2 asses

NA expression

s effect was n

stimulation w

analyzed by

cant, *P < 0.

AP, DDR2, SM

reatment wit

y qRT‐PCR wa

ssed by qRT‐P

n of SNAI1 ass

not additive.

with TGF‐β a

2‐way ANOV

.05, **P < 0.

M22α, Calponi

h TGF‐β and

as non‐signific

PCR was incr

sessed by qRT

All experime

nd/or H2O2 o

VA with com

.01, ***P < 0

n and

H2O2.

cantly

eased

T‐PCR

ents in

over 5

mplete

0.001,

Suppleme

human sa

according

function o

plaque ca

presented

from our

entary Fig. 13

amples obtai

g to standard

of the total n

ap thickness v

d in this analy

original data,

3. EndMT is m

ned at autop

criteria (see

umber of DA

versus % FAP+

ysis. r = 0.614

, a total of 17

more commo

psy from the

methods). Th

API+ cells (per+vWF+ co‐pos

47, p = 0.009.

separate pla

on in plaques

e abdominal

he % of co‐po

field). Limite

itive cells. On

After exclusio

ques from 10

s with thin fib

aorta were c

ositive cells p

ed scatter plo

nly plaques w

on of 7 plaqu

0 patients are

brous cap. Pla

classified as

er 20x field w

ot and regres

with cap thick

ues with cap t

e presented.

aques identif

AHA type V

was expressed

sion line for

kness < 100 µ

thickness > 10

fied in

or VI

d as a

aortic

m are

00 µm

Suppleme

atheroscl

Plaques w

performe

αSMA/vW

as indicat

entary Fig. 1

erosis. AHA t

were classifie

d using vari

WF, (c) SM22α

ed and arrow

14. EndMT g

type V plaqu

ed as AHA t

ious endothe

α/CD31. All s

ws indicate co

gives rise to

es were iden

ype V accor

elial‐VSMC m

cale bars rep

‐positive cells

o infrequent

ntified from h

ding to stan

marker comb

present 50 µm

s.

vascular sm

human aortic

ndard criteria

binations as

m. Insets are

mooth muscl

c samples obt

a (see metho

follows: (a)

shown at hig

e cells in h

tained at aut

ods). Staining

αSMA/CD31

gher magnific

uman

topsy.

g was

1, (b)

cation

Suppleme

function o

SM22α/C

entary Fig. 1

of the total n

D31) or 6 (αS

14 (continued

number of DA

SMA/vWF) dif

d). (d) The %

API+ cells (pe

fferent patien

% of co‐posit

r field). Two

nts were rand

ive cells per

separate pla

domly include

20x field w

aques from 5

ed per analysi

as expressed

(αSMA/CD3

is.

d as a

1 and

Suppleme

cell lines

used in t

control st

entary Fig. 1

from 3 healt

the microarra

aining is show

5. Characteri

hy control su

ay and MMP

wn. Rb rabbit

ization of hu

ubjects for FA

P expression

; Ms mouse.

uman fibrobla

AP, FSP‐1 and

experiments

Scale bars re

ast cell lines

d Vimentin. T

s shown in F

present 100 µ

s. Immunosta

These fibrobla

Figs. 6a ‐ f. C

µm.

aining of fibro

ast lines are

Correspondin

oblast

those

ng IgG

Suppleme

expected

membran

control la

entary Fig. 1

molecular

nes were ima

nes loaded w

6. Original im

weight/size

aged adjacen

with substanti

mmunoblots

is 90kDa. G

t to one ano

ally lower lev

from Fig. 3e

GAPDH expe

other. On the

vels of protein

e. Ladder ma

ected molecu

e right mem

n and not inc

arker sizes sh

ular weight/

branes, the

luded in the m

hown on left

/size is 37kD

first 6 lanes

main Figures.

t. FAP

Da. 2

were

.

Suppleme

expected

membran

additiona

entary Fig. 17

molecular w

nes were ima

l control lane

7. Original im

weight/size

aged adjacen

es and not inc

mmunoblots

is 130kDa.

t to one ano

cluded in the

from Fig. 3g

GAPDH expe

other. On the

main Figures

g. Ladder ma

ected molec

e right mem

.

rker sizes sho

cular weight/

branes, the

own on left.

/size is 37kD

first 6 lanes

CD31

Da. 2

were

Suppleme

expected

final addi

sample un

entary Fig. 18

molecular w

itional lane i

nder conditio

8. Original im

eight/size is 2

s present (m

ons of combin

mmunoblots f

23kDa. GAPD

most rightwar

ned hypoxia a

from Fig. 4m.

DH expected m

rd lane), whi

nd TGF‐β trea

. Ladder mar

molecular we

ich represent

atment.

ker sizes show

eight/size is 3

ted an addit

wn on left. SM

37kDa. Note t

tional sample

M22α

that a

e (4th)

Suppleme

expected

entary Fig. 19

molecular we

9. Original im

eight/size is 1

mmunoblots

130kDa. GAPD

from Fig. 4n

DH expected

n. Ladder ma

molecular we

rker sizes sho

eight/size is 3

own on left.

37kDa.

CD31

Suppleme

on left. A

weight/siz

entary Fig. 20

Activated Cas

ze is 37kDa.

0. Original im

spase 3 expe

mmunoblots f

ected molecu

from Supplem

ular weight/s

mentary Fig.

size is 17kDa

9c. Ladder m

a. GAPDH ex

marker sizes s

xpected mole

shown

ecular

Suppleme

on left. p

52kDa. GA

additiona

entary Fig. 21

pSMAD3 expe

APDH expecte

l control lane

1. Original im

ected molecu

ed molecular

es and not inc

munoblots fr

ular weight/si

r weight/size

cluded in the

rom Supplem

ize is 48kDa.

is 37kDa. On

main Figures

mentary Fig. 1

SMAD3 exp

the right me

.

10g. Ladder m

ected molec

mbranes, the

marker sizes s

ular weight/s

e first 6 lanes

shown

size is

s were

Supplementary Table 1. Complete results of 2‐way ANOVA analyses for Supplementary Fig. 9,

representing comparisons of HUVECs exposed to TGF‐β and/or H2O2.

Figure Event studied

Source of variation (% of total variation, p value)

Bonferroni posttest p value

Inter‐action

H2O2 TGF‐β No TGF‐β vs. TGF‐β

No H2O2 vs. 100µM H2O2


No H2O2

100µM H2O2

200µM H2O2

No TGF‐β

TGF‐β No

TGF‐β TGF‐β

Suppl 9a

Number of cells

0.07%; P = 0.96

89.49%; P < 0.0001

1.15%;P = 0.25

ns ns ns < 0.01 < 0.05 < 0.001 < 0.001

Suppl 9b

BrdU incorporation

1.50%; P = 0.29

91.55%; P < 0.0001

0.37%;P = 0.43

ns ns ns < 0.0001 < 0.001 < 0.0001 < 0.0001

Suppl 9e

% dead cells 0.26%; P = 0.94

57.35%; P = 0.0004

1.49%;P = 0.43

ns ns ns ns ns < 0.01 < 0.01

Supplementary Table 2. List of genes and transcription factors presented in Fig. 3d that are altered in

EndMT/EMT and supporting references.

Abbreviated name

Full name Alternative name

Reference

Endothelial

VWF von Willebrand factor 1

CAV1 Caveolin‐1 2

ICAM2 Intercellular adhesion molecule 2 3

PECAM1 Platelet/endothelial cell adhesion molecule 1

CD31 1

CDH5 Cadherin‐5 VE‐Cadherin; CD144

1

SOX18 SRY‐related HMG‐box 18 4

ENG Endoglin CD105 5

ESAM Endothelial cell adhesion molecule 6

TEK TIE2; CD202B 1

GPR116 G Protein‐Coupled Receptor 1161 7

CD34 CD34 1

FLI1 Friend leukemia virus integration 1 8

EMCN Endomucin 9

TIE1 Tyrosine kinase with immunoglobulin‐like and EGF‐like domains 1

10

LMO2 LIM domain only 2 11

EGFL7 EGF‐like domain‐containing protein 7 Vascular Endothelial‐statin

12

SOX7 SRY (sex determining region Y)‐box 7 13

NOS3 Nitric oxide synthase 3 eNOS 1

KDR Kinase insert domain receptor vascular endothelial growth factor receptor 2

14

LYVE1 Lymphatic vessel endothelial hyaluronan receptor 1

XLKD 15

ETV2 Ets Variant 2 13

SELP P‐selectin CD62 16

CLDN3 Claudin‐3 Tight junction protein

17

VCAM1 Vascular cell adhesion protein 1 CD106 18

SELE E‐selectin CD62E 19

Downregulated in EndMT/EMT

CAV2 Caveolin‐2 20

KRT19 Keratin, type I cytoskeletal 1 cytokeratin‐19 21

TP53 Tumor protein p53 p53 22

MTUS1 Mitochondrial tumor suppressor 1 23

CXADR Coxsackievirus and adenovirus receptor CAR 24

JUP Junction Plakoglobin Desmoplakin‐3; gamma‐catenin

25

DSP Desmoplakin 26

Upregulated in EndMT/EMT

ITGA5 Integrin, alpha 5 CD49e 27

CALD1 Caldesmon 28

MSN Moesin 29

SPOCK1 Sparc/osteonectin, cwcv and kazal‐like domains proteoglycan 1

Testican 30

SPARC Secreted protein acidic and rich in cysteine

Osteonectin 31

ITGAV Integrin alpha‐V CD51 32

AHNAK AHNAK Desmoyokin 33

NOTCH1 34

JAG1 Jagged 1 CD339 35

SLC22A4 Solute carrier family 22, member 4 36

ZEB2 Zinc finger E‐box‐binding homeobox 2 Sip1 37

CXCR4 C‐X‐C chemokine receptor type 4 CD184 38

ZEB1 Zinc finger E‐box‐binding homeobox 1 39

HEY2 Hairy/enhancer‐of‐split related with YRPW motif protein 2

CHF1 39, 40

SNAI1 SNAIL 41

SNAI2 SLUG 41

HEY1 Hairy/enhancer‐of‐split related with YRPW motif protein 1

42

TWIST1 Twist‐related protein 1 TWIST 41

WNT5B Wingless‐type MMTV integration site family, member 5B

43

WNT11 Wingless‐type MMTV integration site family, member 11

44

HEYL Hairy/enhancer‐of‐split related with YRPW motif‐like protein

45

VPS13A Vacuolar protein sorting‐associated protein 13A

46, 47

Mesenchymal

VIM Vimentin 48

SERPINE1 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1

Plasminogen activator inhibitor‐1

49

CTGF Connective tissue growth factor CCN2 50 36

SRGN Serglycin PPG; PRG1 36

TUBA1A Tubulin alpha‐1A chain 36

MYH9 Myosin, heavy chain 9 NMHC‐IIA 51

TPM1 Tropomyosin alpha‐1 chain 52

TAGLN Transgelin SM22; SM22α 41

CDH2 Cadherin‐2 neural cadherin (NCAD)

53

COL5A1 Collagen, type V, alpha 1 54

NT5E 5'‐nucleotidase CD73 55

COL5A2 Collagen, type V, alpha 2 56

SRF Serum response factor 57

ACTA2 Alpha‐actin‐2 alpha smooth muscle actin (αSMA)

41

PLAT Tissue plasminogen activator tPA 58

PLAU Urokinase‐type plasminogen activator 49

CDH11 Cadherin‐11 CAD11 56

P4HA1 Prolyl 4‐hydroxylase subunit alpha‐1 P4HA 59

SERPINE 2 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2

60

PTX3 Pentraxin‐related protein PTX3 56

RECK Reversion‐inducing‐cysteine‐rich protein with kazal motifs

36

FBLN5 Fibulin‐5 56 36

MMP2 Matrix metalloproteinase‐2 61

PLAUR Urokinase receptor CD87 60

PRKCA Protein kinase C alpha PKCα 36

NEXN Nexilin F‐actin binding protein

41


POSTN Periostin Osteoblast specific factor

56

DLC1 Deleted in Liver Cancer 1 STARD12 56 36

COL1A2 Collagen, type I, alpha 2 56

FN1 Fibronectin 52

PLEK2 Pleckstrin‐2 60

COL6A1 Collagen, type VI, alpha 1 62

IGFBP3 Insulin‐like growth factor‐binding protein 3

36

VCAN Versican 63

COL3A1 Collagen, type III, alpha 1 56

NOTCH3 Neurogenic locus notch homolog protein 3

CADASIL 64

CD248 Endosialin; TEM1 65 66

FAP Fibroblast activation protein DPPIV 56

S100A4 S100 calcium‐binding protein A4 67

CNN1 CCN family member 1 Cysteine‐rich angiogenic inducer 61 (CYR61)

68

COL1A1 Collagen, type I, alpha 1 69 36

ADAM12 Disintegrin and metalloproteinase domain‐containing protein 12

70 36

NID2 Nidogen‐2 36

SPP1 Secreted phosphoprotein 1 Osteopontin; OPN 71

CD44 72

FBLN1 Fibulin‐1 56 36

DDR2 Discoidin domain‐containing receptor 2 56


Supplementary Table 3. Complete results of 2‐way ANOVA analyses for Fig. 3e ‐ h, representing

comparisons of HUVECs exposed to TGF‐β and/or H2O2.




Inter‐action




No H2O2

100µM H2O2

200µM H2O2

No TGF‐β

TGF‐β No

TGF‐β TGF‐β

Figure 3e FAP

protein levels

3.4%; P = 0.059

83.50%; P < 0.0001

7.46%; P = 0.0018

< 0.05 ns < 0.05 < 0.05 ns < 0.001 < 0.001

Figure 3f FAP RNA levels

11.65%; P = 0.0012

27.16%; P < 0.0001

33.49%;P < 0.0001

ns < 0.05 < 0.001 ns ns ns < 0.001

Figure 3g CD31 protein levels

8.34%; P = 0.017

73.53%; P < 0.0001

3.99%; P = 0.039

ns ns < 0.01 < 0.001 < 0.01 < 0.001 < 0.001

Figure 3h CD31 RNA levels

9.58%; P = 0.057

35.03%; P = 0.001

0.32%;P = 0.65

ns ns ns < 0.001 ns < 0.001 < 0.05


representing comparisons of HUVECs exposed to TGF‐β and/or H2O2.




Inter‐action




No H2O2

100µM H2O2

200µM H2O2

No TGF‐β

TGF‐β No

TGF‐β TGF‐β

Suppl 10a

Calponin RNA levels

13.85%; P = 0.0020

21.37%; P = 0.0001

33.55%;P < 0.0001

ns ns < 0.001 ns ns ns < 0.001

Suppl 10b

Versican RNA levels

7.95%; P = 0.017

12.47%; P = 0.0023

52.88%;P < 0.0001

< 0.05 < 0.01 < 0.001 ns ns ns < 0.001

Suppl 10c

SNAI2 RNA levels

11.86%; P = 0.0016

36.84%; P < 0.0001

26.77%;P < 0.0001

ns ns < 0.001 ns ns ns < 0.001

Suppl 10d

SNAI1 RNA levels

1.64%; P = 0.34

4.25%; P = 0.071

68.17%;P < 0.0001

< 0.001 < 0.001 < 0.001 ns < 0.05 ns ns

Suppl 10e

SMAD2 RNA levels

0.19%; P = 0.97

0.16%; P = 0.82

8.02%;P = 0.28

ns ns ns ns ns ns ns

Suppl 10f

SMAD3 RNA levels

2.51%; P = 0.31

47.85%; P < 0.0001

19.19%;P = 0.0001

ns ns < 0.01 < 0.05 < 0.01 < 0.001 < 0.001

Suppl 10g

SMAD3 protein levels

13.45%; P = 0.091

26.35%; P = 0.015

19.27%; P = 0.012

ns ns < 0.05 ns ns < 0.01 ns

Suppl 10g

pSMAD3 protein levels

5.33%; P = 0.0076

0.88%; P = 0.36

65.74%; P < 0.0001

< 0.001 < 0.001 < 0.001 < 0.01 ns ns ns


representing comparisons of HCAECs exposed to TGF‐β and/or H2O2.




Inter‐action



No H2O2 200µM H2O2

No TGF‐β TGF‐β

Suppl 12b FAP RNA levels

5.79%; P = 0.080

10.73%;P = 0.021

55.52%;P < 0.0001

< 0.05 < 0.001 ns < 0.05

Suppl 12c DDR2 RNA levels

0.9%; P = 0.54

34.44%;P = 0.0009

19.45%;P = 0.0082

ns < 0.05 < 0.01 ns

Suppl 12d SM22α RNA

levels 17.03%; P = 0.0005

33.44%;P < 0.0001

29.87%;P < 0.0001

ns < 0.001 ns < 0.001

Suppl 12e Calponin RNA levels

7.51%; P = 0.031

13.38%;P = 0.0057

51.12%;P < 0.0001

< 0.05 < 0.001 ns < 0.01

Suppl 12f Versican RNA levels

17.49%; P = 0.0004

23.35%;P < 0.0001

40.08%;P < 0.0001

ns < 0.001 ns < 0.001

Suppl 12g CD31 RNA levels

5.75%; P = 0.25

10.21%;P = 0.13

1.13%;P = 0.61

ns ns ns ns

Suppl 12h TIE2 RNA levels

0.02%; P = 0.95

11.43%;P = 0.12

2.64%;P = 0.44

ns ns ns ns

Suppl 12i SNAI2 RNA

levels 10.97%; P = 0.0064

37.20%;P < 0.0001

28.17%;P < 0.0001

ns < 0.001 ns < 0.001

Suppl 12j SNAI1 RNA

levels 20.10%; P = 0.0099

4.70%;P = 0.18

25.63%;P = 0.0043

< 0.001 ns < 0.05 ns

Supplementary Table 6. PCR primers.

Gene Primers Length of

PCR products (base pairs)

Forward Reverse

Mouse Genotyping Primers

ApoE GCCTAGCCGAGGGAGAGCCG (WT) TGTGACTTGGGAGCTCTGCAGC (Mutant) GCCGCCCCGACTGCATCT

Mutant: 245 WT: 155

Cre ATTGCTGTCACTTGGTCGTGG

GAAAATGCTTCTGTCCGTTTGC 200

YFP AAAGTCGCTCTGAGTTGTTAT

(WT) GGAGCGGGAGAAATGGATATG (Mutant) GCGAAGAGTTTGTCCTCAACC

Mutant: 320 WT: 600

Human Primers

18S rRNA TTTCGGAACTGAGGCCATGA GCAAATGCTTTCGCTCTGGTC 110

FAP CAAGTGGCAAGTGGGAGGCCA TGGGGATGCCTGGGCCGTAG 246

DDR2 GACTTGCACACCCTCCATTT GAGTGGTCGGTGACTGGAAT 251

SM22α CCTGGCTAGGGAAACCCACCCT TCTGGGGAAAGCTCCTTGGAAGT 300

Calponin GGCCAGCATGGCGAAGACGAAA TGTGCCCAGCTTGGGGTCGT 205

CD31 GCGAGTCATGGCCCGAAGGC GGTGGTGCTGACATCCGCGA 246

SNAI1 GCACGGCCTAGCGAGTGGTT GGGCTGCTGGAAGGTAAACTCTGG 171

SNAI2 AACTCACACGGGGGAGAAGCCT CAGTGTGCTACACAGCAGCCAGA 192

SMAD2 AGTGCCCCGACACACCGAGA TCTGCTGGAGAGCCTGTGTCC 207

SMAD3 TCAAGAAGACGGGGCAGCTGGA GCACCAACACAGGAGGTAGAACTGG 297

TIE2 GCAATGAAGCATGCCACCCTGG GGTAGCGGCCAGCCAGAAGC 241

Versican GGTGCACTTTGTGAGCAAGA TTCGTGAGACAGGATGCTTG 159

Ve‐Cadherin AACTTCCCCTTCTTCACCC AAAGGCTGCTGGAAAATG 368

eNOS GTGGCTGTCTGCATGGACCT CCACGATGGTGACTTTGGCT 121

KDR TGCCTACCTCACCTGTTTC GGCTCTTTCGCTTACTGTTC 114

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