Exosomes Mediate the Cytoprotective Action of

DOI: 10.1161/CIRCULATIONAHA.112.114173
Exosomes Mediate the Cytoprotective Action of Mesenchymal Stromal Cells
on Hypoxia-Induced Pulmonary Hypertension
Running title: Lee et al.; Exosomes as paracrine vectors of MSC action
Changjin Lee, PhD1,2; S. Alex Mitsialis, PhD1,2*; Muhammad Aslam, MD1,2; Sally H. Vitali,
MD3,4; Eleni Vergadi, MD1,5; Georgios Konstantinou, MD1; Konstantinos Sdrimas, MD1;
Angeles Fernandez-Gonzalez, PhD1,2; Stella Kourembanas, MD1,2*
*
1
Equal contributors to this work as senior authors
Div off Newborn
New
wbo
b rn
n Medicine;
Med
edic
i in
ic
ne; 3Di
Div
D
v of Critical
Cri
riti
tica
ti
cal Care
Ca Medicine,
Med
edic
icin
ic
ne, Ch
Chil
Children’s
ildr
dren
dr
e ’ss H
en
Hospital
ospi
os
pita
pi
tall Boston;
Bost
Bo
stton
on;;
2
Dept
p off P
Pediatrics;
ediaatrrics;; 4D
Dept
ept
pt ooff An
Anaest
Anaesthesia,
theesiaa, H
Harvard
arrvaard M
Medical
eddicaal Sc
School,
cho
ooll, B
Boston,
ostoon,, MA
MA;
5
Present
Pres
Pr
esen
es
en
nt Ad
Addr
Address:
d esss: U
dr
University
niive
vers
rsiity
rs
ity of C
Crete,
rete
re
tee, He
Hera
Heraklion,
r kl
ra
klio
ion,
n C
n,
Crete,
rete
re
te,, Gr
Greece
ree
eece
ce
Address
Add
Ad
dress for
for Correspondence:
Correspondence:
d
Stella Kourembanas, MD
Division of Newborn Medicine
Children’s Hospital Boston
300 Longwood Avenue
Boston, MA 02115
Tel: 617-919-2355
Fax: 617-730-0260
E-mail: stella.kourembanas@childrens.harvard.edu
Journal Subject Codes: [18] Pulmonary circulation and disease; [130] Animal models of
human disease; [147] Growth factors/cytokines
1
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DOI: 10.1161/CIRCULATIONAHA.112.114173
Abstract:
Background—Hypoxia induces an inflammatory response in the lung manifested by alternative
activation of macrophages with elevation of pro-inflammatory mediators that are critical for the
later development of hypoxic pulmonary hypertension (HPH). Mesenchymal stromal cell (MSC)
transplantation inhibits lung inflammation, vascular remodeling and right heart failure, and
reverses HPH in experimental models of disease. In this study, we aimed to investigate the
paracrine mechanisms by which MSCs are protective in HPH.
Methods and Results—We fractionated mouse MSC-conditioned media to identify the
biologically-active component affecting in vivo hypoxic signaling and determined that exosomes,
secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and
y and ppro-proliferative
p
g monocyte
y
the induction of ppro-inflammatory
mediators,, including
chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murinee model
mo
odeel of HPH.
HPH
PH.
ntravenous delivery of MSC-derived exosomes (MEX) inhibited vascular remodeling and HPH,
Intravenous
whereas MEX-depleted
MEX-depl
p eted media or fibroblast-derived exosomes had no effect. MEX suppressed
he hypoxic
hyppoxi
hy
xicc activation
actiiva
vati
tion
o of
of signal
sign
si
g al transducer
tran
nsdduccer
e andd activator
actiivatorr ooff transc
sccript
ptio
ionn 3 (S
STA
AT3)) an
andd th
tthee
the
transcription
(STAT3)
uupregulation
pre
regu
eg lation ooff tthe
he mi
miRR--17 ssuperfamily
uper
up
erfa
f mi
fa
m ly of microR
m
i RNA cclusters,
lusster
lu
st rs, w
hereaas itt in
here
ncrea
creassedd lu
lungg llevels
ev
vels
miR-17
microRNA
whereas
increased
off miR-204,
miR
iR-204,, a key
key microRNA
miccrooRNA
mi
oRNA
A whose
who
hosse expression
expres
exp
presssiion is
is decreased
deccre
de
crease
easeed inn human
huuman
uman
n PH.
PH.. MEX
MEX produced
prodduced
uced
d by
by
huma
mann umbilical
um
mbi
b lica
cal co
cord
rd M
SCss in
SC
inhi
hibi
bited ST
STAT
AT33 si
signal
lin
ingg in iisolated
solate
so
tedd hu
huma
mann pu
pulm
lmonar
aryy artery
arrte
tery
ry
human
MSCs
inhibited
STAT3
signaling
human
pulmonary
endothelial cells
cell
ce
llss de
ll
demo
mons
mo
n tr
ns
trat
atin
at
ingg a di
in
dire
rect
ctt effect
eff
ffec
e t of MEX
ec
MEX on
on hypoxic
hypo
hy
poxi
po
xic vasc
vvascular
asc
scul
ular
ul
ar ccells.
ells
el
l.
ls
demonstrating
direct
Conclusions—This study indicates that MEX exert a pleiotropic protective effect on the lung
and inhibit PH through suppression of hyperproliferative pathways, including STAT-3 mediated
signaling induced by hypoxia.
Key words: hypertension, pulmonary; inflammation; hypoxia; signal transduction
2
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DOI: 10.1161/CIRCULATIONAHA.112.114173
The lung’s response to low levels of environmental oxygen is multifactorial. Diverse signaling
pathways, activated or impaired by alveolar hypoxia, converge on endothelial and vascular
smooth muscle cells to perturb pulmonary vascular homeostasis. Chronic hypoxia results in
pulmonary vascular remodeling, a key pathological feature of pulmonary hypertension (PH).
Inflammation plays a prominent detrimental role in most types of human PH and also in animal
models of the disease, such as the monocrotaline- and hypoxia-induced PH (HPH) in rodents.
The early component of hypoxia-induced lung inflammation, peaking during the first two to
three days of hypoxic exposure1, is characterized by alternative activation of alveolar
macrophages and appears to be causal to the subsequent vascular remodeling and the
development of HPH2.
Despite the significant progress in our understanding of the pathophysiology of PH as
well
we
ll as
as the
the treatment
treeatm
tr
ment
en of its symptoms, there is noo cure
cuure for this disease
dissea
e see and
and no single therapy has
bbeen
een
n proven ef
effe
effective.
fecttiv
fe
ve. G
Given
iven tthe
iven
he ccomplex
he
omppleex ppathways
om
athwaays in
involved
nvolv
nvo
olved in th
the
he ppathogenesis
he
athhogeene
nesi
siss off P
PH,
H
H,
therapies
her
erap
apie
ap
i s aimed
ie
aime
ai
m d at more
me
mor
oree than
th n one
one pathway
pat
athw
hw
way and
andd perhaps
perh
hap
apss more
morre than
mo
tha
hann one
one cellular
cell
ce
llul
ullar
a target
tarrge
gett may
mayy prove
prrov
ve
too be more efficacious.
efffic
icac
accio
ious
us.. Stem
us
S em cell-based
St
cellll ba
base
s d therapeutic
th
herrap
peu
e ti
t c approaches
appr
ap
prroa
oach
ches
ch
es hhold
olld su
such
ch a ppromise
romi
ro
mise
mi
se as
as they may
y
simultaneously target multiple signaling pathways and have long lasting effects. The therapeutic
potential of mesenchymal stromal cells (MSCs; also referred to as mesenchymal stem cells or
multipotent stromal cells) derived from the bone marrow, adipose, and other tissues, has been
recognized in several animal models of lung disease3. In models of pro-inflammatory lung
diseases, such as bleomycin or endotoxin-induced lung injuries4-6 as well as the neonatal murine
model of bronchopulmonary dysplasia (BPD)7, 8, MSC delivery ameliorated lung injury,
decreased lung inflammation and fibrosis, and increased survival. MSC delivery was reported to
inhibit PH induced by monocrotaline in the rat9 and HPH in the mouse10. However, although a
3
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DOI: 10.1161/CIRCULATIONAHA.112.114173
robust protection against lung injury upon MSC treatment was observed in most of the above
animal models, only a small fraction of donor cells were retained in the recipient lung. This
observation suggested that engraftment and direct tissue repair was not the sole mechanism of
MSC therapeutic function and paracrine mechanisms were contemplated. In support of this, we
observed that injections with culture media conditioned by MSCs can efficiently inhibit
parenchymal injury, vascular remodeling and right ventricular hypertrophy (RVH), completely
supplanting MSC treatment on the neonatal murine model of BPD7, 11. In vitro experiments
demonstrated anti-proliferative properties of the MSC secretome on pulmonary vascular smooth
muscle cells10, again suggesting that MSC paracrine factors can play a major role in preventing
lung
ung injury and vascular remodeling. Concordant with our observations, paracrin
paracrine
ne aand
ne
ndd
immunomodulatory
mmunomodulatory paradigms have been recently proposed to account for enhanced MSC
therapeutic
herrap
apeeuti
euti
ticc fu
func
function
cti
tioon
on in the context of a number of ddisease
isease modelss12.
We ha
have
ave pre
previously
revi
viou
vi
ouusl
slyy pe
perf
performed
rfor
orrmed
med pproteomic
roteeom
eomic aanalysis
nalys
ysis
ys
is ooff MS
MSC-conditioned
SC-co
C-cond
n itio
nd
io
one
nedd m
medium
edi
d um
di
m7, wh
w
which
ich
in
n aaddition
ddit
dd
i io
it
ionn to iimmunomodulatory
mm
mun
unom
omod
om
odullat
a or
oryy fa
fact
factors,
ctor
ors,
or
s, rrevealed
e ea
ev
eale
leed th
tthee pr
pres
presence
esen
es
nce
c ooff a num
nnumber
umber
mber ooff pr
pro
proteins
ot ins
otei
ns
including
ncluding CD63,
CD
D63
63,, CD81,
CD81
CD
81,, moesin,
moes
mo
esin
es
n, lactadherin
l ct
la
ctad
ad
dhe
heri
r n (MFGE8),
ri
(MFG
(M
FGE8
FG
E ),, hheat-shock
E8
eatea
t shhoc
tockk pr
prot
protein
otei
ot
einn 90
ei
9 ((hsp90),
hsp9
hs
p90)
p9
0), and
0)
hsp70, reported to be associated with secreted vesicles known as exosomes13, 14. Secreted
membrane microvesicles, especially the better-defined subclass represented by exosomes15, have
been recognized as important mediators of cell-to-cell communication and as participants in
immunomodulatory mechanisms16. Exosomes are small heterogeneous microvesicles, 30~100
nm in diameter, that are stored within multivesicular bodies (MVB) and released into the
environment upon fusion of the MVB with the plasma membrane. Exosomes and microvesicles
have been isolated and characterized from various cell types including dendritic cells17,
macrophages18, tumor cells19, and embryonic stem cells20 and information is rapidly
4
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DOI: 10.1161/CIRCULATIONAHA.112.114173
accumulating on their diverse biological function and their cell type-specific molecular
composition. The physiologic relevance of MSC-derived exosomes (MEX) has not yet been
evaluated in lung diseases, even though their cellular origin and the recently recognized
paracrine function of MSCs may imply a promising therapeutic potential for secreted
microvesicles in lung injury.
To address the above questions, we fractionated MSC-conditioned media (CM) through
size-exclusion chromatography to identify the biologically-active component protecting against
hypoxia-induced lung inflammation and HPH. Using the murine model of HPH, we demonstrate
here that MEX are the critical vectors of MSC action: MEX delivery in vivo suppressed HPH and
vascular remodeling. Moreover, pro-proliferative pathways were also blocked bby
y ME
MEX
X
treatment,
reatment, as evidenced by the suppression of signal transducer and activator of transcription
(STAT3)
phosphorylation
miR-204,
STA
TAT3
T3)) ph
T3
pho
osphhor
oryl
y ation resulting in increased
d llung
unng levels off m
iR
R-204
04,, a microRNA enriched
04
in
experimental
n distal
dis
istal pulmonary
pulm
mon
onar
a y arterioles
ar
arrteri
riol
ri
oles
ol
es that
thatt iiss ddown-regulated
own-rreg
gulaated inn both
both
h hu
hhuman
mann PH aand
ma
nd
d iin
n ex
exp
peri
peri
rim
menntal
ntall
models
disease
mo
ode
dels
ls ooff di
dis
sease2211. We found
seas
foun
fo
unnd that
that hypoxia
hypox
ypox
oxia
ia upregulates
upr
p egu
egulat
attess members
memb
memb
mbeers
ers of
o the
the miR-17
miR
iR-1
- 7 fa
-1
fami
family
milly
ly ooff
microRNA cclusters
lu
ust
ster
errs in lung
lun
u g tissue,
tiiss
s ue
ue,, microRNAs
m crroR
mi
oRNA
NAss shown
NA
shhow
ownn too bbee un
unde
derr th
de
thee re
regu
gula
gu
lato
la
to
ory ccontrol
ontr
on
t ol of
tr
under
regulatory
STAT3, and show that MEX treatment efficiently suppresses this pro-proliferative signal.
Combined, our findings point to MEX as the key effectors of MSC paracrine function with the
potential to serve as vehicles of lung-targeted therapy.
Methods
Animal model and hypoxic exposure.
The HPH mouse model has been well-established and used by our group extensively in
previously-published work 1, 2, 22. The hypoxic exposure and treatment protocols used in this
5
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DOI: 10.1161/CIRCULATIONAHA.112.114173
study are described in the Supplemental Material. All animal experiments were approved by the
Boston Children’s Hospital Animal Care and Use Committee.
Preparation of exosomes
Isolation of mouse bone marrow-derived MSCs and MSCs from human umbilical cord
Wharton’s Jelly (hUC-MSCs) followed by immunoselection (Supplemental Figure 1) and
collection of conditioned media (CM) is outlined in the Supplemental Material.
Concentrated conditioned media were applied on a column of 16/60 Hiprep Sephacryl S400 HR (GE Healthcare, Piscataway, NJ) that was pre-equilibrated with a buffer containing 20
mM sodium phosphate (pH 7.4) and 300 mM NaCl using an ÄKTA purifier liquid
chromatography system (GE Healthcare, Piscataway, NJ). Fractions (1 ml) weree co
collected
oll
l eccte
tedd at a
flow rate of 0.5 ml/min. Polystyrene nanospheres of 50 nm diameter (Phosphorex, Fall River,
MA)
fractions
corresponding
MA
A) were
weree uused
seed as a size reference and elution fr
rac
a tions correspo
ond
n in
ng to this standard’s
retention
analyzed.
eteention volume
volu
umee were
wer
eree pooled
pool
pool
oled
ed
d and
andd further
furt
urthe
ther an
nalyzeed.
nal
For
isolation
fibroblasts,
serum-free
For th
thee is
sollat
atio
io
on of eexosomes
xoso
some
mess from
me
from hhUC-MSCs
UC-M
UC
-M
MSC
SCss aand
nd hhuman
uman
um
an
n ddermal
errma
mall fi
fibr
brrob
obla
lassts,
la
sts, se
erum
erum
m-ffre
r e
culture medium
filtered
(0.2
concentrated
mediium cconditioned
ondi
on
d ti
di
t on
o ed ffor
orr 224
4 ho
hhours
urss wa
ur
wass fi
filt
ltterred (0.
0.22 μm
0.
μm)) an
aand
d co
conc
ncen
nc
entr
en
t atted bby
tr
y
ultrafiltration device with 100 kDa cut-off (Millipore). Exosomes in CM were precipitated with
1/3 volume of polyethylene glycol (PEG) buffer (33.4% PEG 4000, 50 mM HEPES (pH 7.4), 1
M NaCl) overnight at 4°C followed by centrifugation at 12,000 xg for 5 min and resuspension in
PBS (pH7.4). Exosomes in PEG-precipitated fraction were further purified by S200 sizeexclusion chromatography. Seventy-five μl sample was applied on a S200 column (Clontech,
Mountain View, CA) preequilibrated with PBS by spinning at 700 xg for 5 min and the exosomal
fraction was subsequently eluted in the flow-through by centrifugation at 700 xg for 5 min.
In some experiments, exosomes were isolated by ultracentrifugation at 100,000 xg for 2
6
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DOI: 10.1161/CIRCULATIONAHA.112.114173
hours and the pellet was subsequently washed with PBS followed by repeat ultracentrifugation
for 2 hours at the same speed. Exosome pellet resuspended in PBS was measured for protein
concentration by Bradford assay (Bio-Rad, Hercules, CA). Expression of exosomal markers
between the two preparations was similar, as shown in Supplemental Figure 2.
Statistical Analysis
All values are expressed as mean ± standard deviation (SD). All comparisons between
experimental and control groups were performed by One-way ANOVA (analysis of variance)
with Tukey-Kramer post-test using PRISM 5 statistical software (GraphPad software, San
Diego, CA) unless otherwise indicated. A value of p < 0.05 was considered to indicate
statistically
groups.
tatistically significant differences. Student’s t-test was used to compare two grou
ou
upss.
See Supplemental Material for a detailed description of further experimental methods.
Results
R
essults
su
Factors
Fa
act
c or
orss secreted
secr
se
cret
cr
eted
ed
d by
by MSCs
MSC can
can
n prevent
pre
reve
veentt hypoxia-induced
hypox
ypox
oxia
ia
a-iind
ducced pulmonary
pul
u mo
mona
n ry inflammation.
na
inffla
l mm
mmat
atiion
at
n.
To determinee if
if hypoxic
hypo
hy
poxi
po
x c lung
xi
lu
ung inflammation
inf
nfla
nf
lamm
la
m at
mm
atio
io
on responds
reesppon
onds
ds too MSC
MSC paracrine
par
araccri
r ne signals,
sig
igna
n ls
na
ls,, we injected
inj
n ected micee
with concentrated serum-free culture media conditioned by either mouse MSCs (MSC-CM) or
by mouse lung fibroblasts (MLF-CM) and exposed the animals to normobaric hypoxia (8.5% O2)
for 48 hours. In the control group injected with vehicle (serum-free culture media), hypoxia
resulted in pulmonary influx of macrophages, as assessed in bronchoalveolar lavage fluid
(BALF) and this response was blocked in animals treated with MSC-CM but not in the group
treated with MLF-CM (Figure 1A). We also assessed, in cell-free BALF, levels of monocyte
chemoattractant protein-1 (MCP-1), a cytokine transiently upregulated in the lung by early
hypoxia2, 23 and levels of hypoxia-induced mitogenic factor (HIMF), a pleiotropic factor with
7
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DOI: 10.1161/CIRCULATIONAHA.112.114173
pro-inflammatory, mitogenic and chemokine-like properties24. In animals injected with either
vehicle or MLF-CM, BALF levels of both MCP-1 and HIMF were highly increased by hypoxia
and this increase was effectively suppressed by MSC-CM treatment (Figure 1B). These results
indicate that, as we have previously reported on the model of hyperoxia-induced BPD7, 11, the
protective effects of MSC treatment in HPH10 involve mainly paracrine mechanisms.
The anti-inflammatory activity in MSC-CM is associated with exosomes
In order to identify the biologically-active component of MSC-CM, we fractionated concentrated
conditioned media through size-exclusion chromatography. Polystyrene nanospheres of 50 nm
diameter served as a hydrodynamic radius standard to identify the exosomal fraction, and
of the
thee standard
sta
tand
ndar
nd
ard
fractions in a protein peak eluting with a retention volume corresponding to thatt of
were pooled (Figure 2A, Fraction I). Negative staining electron microscopic analysis revealed
Frrac
acti
tioon
ti
on I to
to contain
cont
nttain
ain heterogeneous microvesicles
es tthat
hat were absent
n inn fractions
nt
fraactions
fr
ac
corresponding to
Fraction
he retention
re
volu
vo
l me
lu
me ooff mo
moie
ieti
ie
ties
ti
es ooff sm
maller ssize
izze (Fi
Figure
Fi
re 22B,
B, Fr
Fra
acttion II).
II)). Fr
raccti
tion
onn I w
as hhighly
as
ig
ighl
ghl
hlyy
the
volume
moieties
smaller
(Figure
Fraction
Fraction
was
en
nri
rich
ch
hed
e iin
n microvesicles
m cr
mi
crov
ovves
esiiclees 30
0-1
- 00 nnm
m iin
n ddiameter
iame
ia
metter ex
me
xhiibit
ibittin
ingg bi
bico
co
onccav
a e morp
m
orp
phoolo
logy
gy,, a di
gy
ist
stin
in
nct
enriched
30-100
exhibiting
biconcave
morphology,
distinct
morphologica
caal fe
ffeature
atur
at
u e of
ur
o eexosomes
xoso
xo
s me
so
mess ((Fig
Fiig 2C
2C,, ar
arro
rows
ro
ws).
ws
) Ex
).
Exos
osom
os
omes
om
es w
eree pr
er
pres
esen
es
entt in bboth
othh MSC- andd
ot
morphological
arrows).
Exosomes
were
present
MLF-CM and, in this report, MSC exosome preparations are termed MEX, whereas MLF
exosome preparations are termed FEX. Both MEX and FEX contain diverse mature microRNAs
(see below) and also Dicer (Figure 2D), a component of the cytoplasmic microRNA maturation
complex. However, relative abundance of each exosomal marker differs depending on the
cellular origin of the microvesicles.
MEX preparations were efficacious in suppressing hypoxic inflammation when injected
into animals, whereas the MSC-CM fraction depleted of exosomes (ExD-CM) had no significant
effect and FEX had a partial inhibiting effect (Figure 2E). Concordantly, levels of pro-
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DOI: 10.1161/CIRCULATIONAHA.112.114173
inflammatory mediators in cell-free BALF of hypoxic animals were suppressed only by MEX
but not by FEX or ExD-CM treatment (Figure 2F), indicating that the ability to suppress early
hypoxia-induced pulmonary inflammation is associated specifically with exosomes of MSC
origin.
Dose response effects of MEX on lung inflammation
We have previously reported that suppression of the entire period of the inflammatory response
to early hypoxia is required to protect animals from later development of PH 2. This
inflammatory response is transient in the murine model, peaking within 2-3 days of hypoxic
exposure and subsiding by day 7 (Figure 3A, left panel). We therefore assessed the effect of
MEX treatment on the temporal profile of hypoxic lung inflammation, and we fo
foun
undd th
un
that
at a llow
ow
found
dose of MEX (0.1 μg/animal, via jugular vein) was able to delay but not to completely suppress
he pu
pulm
lmon
lm
onar
on
aryy in
nfl
fluux of macrophages, resulting iin
n a shift of the inflammatory
inf
n lamm
nf
mmaatory peak towards later
mm
the
pulmonary
influx
imes
mees (Figuree 33A,
A, mi
idd
ddle
le ppanel).
aneel).
an
el). T
h ob
he
bserrveed te
empooral
al pprofile
rofi
ro
file
lee ooff pu
pulm
lmon
lm
onar
aryy ma
ar
acrropphaage
times
middle
The
observed
temporal
pulmonary
macrophage
nfl
flux
ux w
as pparalleled
aralllelled
aral
led by tthe
h ttemporal
he
em
mpo
pora
rall prof
pprofile
rof
ofil
ilee off iinduction
il
nduction
ndu
tion ooff th
thee pr
proo-in
oin
nflaamma
amma
mato
t ry m
to
arrkeers
rs,,
influx
was
pro-inflammatory
markers,
MCP-1, inter
rle
leuk
u in
uk
in-6
-6 ((IL-6),
I -6
IL
6), G
alec
al
e ti
ec
tinn-3,
3 aand
3,
nd H
IM
MF, in ccell-free
ellel
l fr
lfree
ee B
ALF,
AL
F, w
h ch
hi
h aalso
lsoo sh
ls
shifted to a
interleukin-6
Galectin-3,
HIMF,
BALF,
which
later time (4 to 7 days, Figure 3B). In contrast, a treatment consisting of two sequential
injections of MEX, one prior to exposure to hypoxia and a second injection at day 4, just prior to
the delayed inflammatory peak (Figure 3A, middle panel), efficiently suppressed pulmonary
influx of macrophages over the entire period of inflammatory responses to early hypoxia (Figure
3A, right panel). However the peak of pro-inflammatory markers in BALF, although delayed by
the first dose, was not affected by the second injection of MEX (Figure 3B).
Multiple administrations of low doses of MEX, but not FEX, ameliorate pulmonary
hypertension, right ventricular hypertrophy, and lung vascular remodeling
9
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DOI: 10.1161/CIRCULATIONAHA.112.114173
The physiologic consequences of partial or complete abrogation of the early inflammatory
response to hypoxia are seen in Figure 3C and 3D. A single low dose of MEX did not protect
against the elevation of RVSP or the development of RVH after three weeks of hypoxic
exposure, whereas the double injection regimen significantly improved both variables. These
results mirror the physiologic response we had observed using pulses of heme oxygenase-1 (HO1) overexpression to completely or partially suppress the early hypoxic lung inflammation 2 and
suggest a dose- and time-sensitive window for anti-inflammatory treatments to confer protection
from HPH. Importantly, FEX treatment using the double injection protocol did not have any
physiologic effect, buttressing the assertion that the function(s) protecting against HPH reside
pecifically with exosomes produced by MSCs. Furthermore, animals treated w
ithh tw
it
twoo do
dose
sess of
se
specifically
with
doses
MEX and exposed to three weeks of hypoxia did nott develop vascular remodeling as determined
Į-ssmoot
moot
oth
h mu
musc
scle
sc
le actin (Į–SMA) staining, wh
her
e eas the same tr
rea
e tm
men
entt protocol with FEX
byy Į-smooth
muscle
whereas
treatment
esuult
l ed in me
edi
dial
a w
all hy
al
hype
pert
rtrroph
rt
rophhy si
simi
milar to veh
hiccle--tr
-treat
reatted
d ccontrols
onttrol
trolss (F
Figu
gu
ure
re 44).
).
resulted
medial
wall
hypertrophy
similar
vehicle-treated
(Figure
sing
ngle
ng
le h
igh
ig
h do
osee M
EX
X ttreatment
reeatm
tmen
entt in
en
nhi
hibi
bits
bi
ts h
ypooxicc iinflammation,
yp
nfla
nf
lamm
la
mmat
mm
atio
ion,
io
n,, va
ascu
ascu
ula
ar re
rem
mode
mode
deli
ling
ng aand
n
nd
A si
single
high
dose
MEX
inhibits
hypoxic
vascular
remodeling
HPH.
The incomplete protection from HPH by two sequential low doses of MEX could be related to
the failure in completely suppressing early hypoxic inflammation. To test the efficacy of higher
MEX dosages on early hypoxic inflammation and HPH, 10 μg MEX were injected through the
tail vein and mice were exposed to hypoxia for 2 and 7 days. A higher dose of MEX prevented
pulmonary influx of macrophages similarly with two sequential injections of MEX (Figure 5A)
and importantly, also completely abrogated the elevation of pro-inflammatory marker FIZZ1/HIMF in the lung for the entire period of early hypoxic responses. An equivalent dose of FEX
had no effect (Figure 5B). Next, to examine the efficacy of higher dose of MEX on vascular
10
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DOI: 10.1161/CIRCULATIONAHA.112.114173
remodeling and HPH, 10 μg of MEX were injected and mice were exposed to chronic hypoxia
for three weeks. Compared to vehicle-injected (PBS) controls, the MEX-treated group had
significantly (p < 0.001, One-way ANOVA) decreased RVSP (Figure 6A) and did not develop
RVH in response to chronic hypoxia (Figure 6B). MEX treatment prevented pulmonary vascular
remodeling, as assessed by D-SMA staining (Figure 6C). Morphometric analyses on small
arterioles revealed a significant effect on the medial wall thickness index, with values in the
MEX-treated group approximating those of the minimally-muscularized normoxic vessels
(Figure 6D). Taken together, the above results strongly suggest that the protective mechanism
of MEX action is through blocking inflammatory lung responses to early hypoxia, which when
left
eft unchecked, activate pro-proliferative pathways in the vascular wall thereby in
iincreasing
ncr
creaasi
cr
sing
ngg
medial wall thickness and altering vascular cell phenotype.
MEX
STAT3
ME
EX inhibit
inhi
inhi
hibi
bitt ST
bi
TAT3
AT activation by hypoxia
Early
STAT3
the
mouse
E
arl
rlly hypoxiaa resulted
reesuultted
d in
in activation
acttiv
ac
tivat
atio
io
on off S
TA
AT3
3 in th
he mo
mous
usee lung
lung
g tthrough
hrou
ou
ughh pphosphorylation
hosp
ho
spho
hory
ho
ry
yla
lati
t on
on aatt
Tyr-705
without
total
STAT3
This
activation
was
Ty
Tyrr-70
r7055 an
70
and
d wi
with
thou
th
outt any
ou
any ef
eeffect
fect
fe
c oon
ct
n the
the to
tota
tall llevels
ta
eve
vels
ve
lss ooff STAT
S
TAT
AT33 pr
pprotein.
otei
ot
eiin.
n T
hiis ac
ctiiva
vati
tioon
ti
on w
ass
suppressed
treatment,
FEX
STAT3
transcription
efficiently su
upp
ppre
ress
re
ssed
ss
ed bby
y ME
MEX
X tr
trea
eatm
ea
t en
tm
ent,
t bbut
t,
ut nnot
ot F
E ((Figure
EX
Figu
Fi
gure
gu
re 77A).
A). S
TAT3
TA
T iiss a tr
T3
tran
ansc
an
s ription
factor integral to signaling pathways of many cytokines and growth factors and its activation
plays a critical role in respiratory epithelial inflammatory responses25, 26. Importantly, persistent
ex vivo STAT3 activation, has been linked to the hyperproliferative and apoptosis-resistant
phenotype observed in pulmonary artery endothelial cells (PAECs) 27 and pulmonary artery
smooth muscle cells (PASMCs)28 from patients with idiopathic pulmonary arterial hypertension
(IPAH). Therefore, to determine whether MEX regulate STAT3 activation on lung vascular
cells, we exposed primary human PAECs (hPAECs) to hypoxia and assessed pY-STAT3 levels.
As depicted in Figure 7B, exposure of hPAECs to hypoxia results in robust activation of STAT3
11
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DOI: 10.1161/CIRCULATIONAHA.112.114173
by Tyr-705 phosphorylation. Treatment with mouse MEX or MEX derived from MSCs isolated
from human umbilical cord stroma29 completely abrogated this response. In contrast, neither
mouse FEX, human FEX, nor the fraction of human umbilical cord MSC-CM depleted of
microvesicles (hUC-ExD-CM) had any effect. Besides demonstrating that suppression of STAT3
activation is a property shared by MEX of both human and mouse origin, these results strongly
suggest that direct suppression of hypoxic signaling in pulmonary vascular cells is a primary
function underlying the protection conferred by MEX treatment. In concordance, we found that
MEX dose-dependently inhibited PASMC proliferation rate in response to serum-derived
mitogens (Supplemental Figure 3), confirming that MEX have direct effects on lung vascular
cells.
Differential miRNA content in MEX vs. FEX
numb
nu
mber
mb
err ooff recent
rece
ceent
nt studies report the successfull horizontal
horrizontal transfer
ho
transfe
fer off functional
fe
functional
un
A number
mRNA and
0, 31
1
miR
RN specie
RNA
es from
f om
fr
om exosomes
exo
xoso
some
so
mess into
me
in
nto
o recipient
reeci
ecipieent
nt cells
cellls330,
. To eevaluate
valu
va
lu
uate po
potential
oteent
ntia
iall di
ia
diff
differential
ffer
ff
erren
e ti
t al ssignals
ignnals
ig
miRNA
species
released
ele
leas
assed
e bby
y ME
M
MEX
X vs
vs F
FEX
EX
X tthat
haat co
coul
could
uldd medi
ul
m
mediate
edi
diat
atee th
at
thei
their
eirr ttherapeutic
herrap
peu
euti
t c ef
effects
ffe
fect
ctss in
ct
in vivo,
viv
ivoo, we
w qquantified
uant
nttiffie
i d
relative
elative levels
levells of
o a nnumber
um
mbe
b r of can
candidate
andi
an
d da
di
d tee m
miRNAs
iR
RNA
NAss in
nM
MEX
EX aand
nd F
FEX
EX ppreparations.
repa
re
para
pa
rati
ra
tion
ons.
on
s We
s.
W found thatt
relative to FEX, MEX contain significantly increased levels of miRNA-16 and miRNA-21.
Interestingly, although let7b miRNA levels were comparable within these two types of
exosomes, let7b pre-miRNA was significantly enriched in MEX vs FEX (>10 fold)
(Supplemental Figure 4). These findings point to distinct and potentially important microRNAmediated regulatory signals delivered to the lung by MSC-derived exosomes.
MEX treatment suppresses the hypoxic induction of the miR-17 microRNA superfamily
and increases levels of anti-proliferative miR-204 in the lung.
STAT3 (activated by either vascular endothelial growth factor or IL-6) has been reported to
12
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DOI: 10.1161/CIRCULATIONAHA.112.114173
directly regulate the transcription of the miR-17~92 cluster of microRNAs in PAECs, resulting
in decreased levels of bone morphogenetic protein receptor-2 (BMPR2), a target of miR-1732.
Therefore, we assessed the effect of hypoxia and MEX treatment on the miR-17~92 cluster of
microRNAs and its conserved paralog clusters, miR-106b~25 and miR-106a~363. These
microRNA clusters have been postulated to be pro-proliferative and to target an array of genes
involved in the G1/S phase transition33. We found that select microRNAs representing all three
clusters of the miR-17 superfamily were upregulated by hypoxia in the lung, and this
transcriptional activation was efficiently suppressed by MEX treatment (Figure 8A).
Interestingly, levels of microRNAs involved in hypoxic signaling networks, such as miR-199a5p, a microRNA reported to stabilize HIF1Į in cardiac myocytes34, miR-214, whi
which
hich
hi
ch
h sha
shares
hare
ha
ress th
re
thee
same
ame host gene with miR-199 35, or miR-210, a hypoxamir under direct hypoxia-inducible
fa
acttor
or-1
-1
1 ((HIF1Į
HIF1
HI
F Į rregulation
egu ation36, were not affected by M
egul
MEX
EX treatment
nt (Fi
(Figure
Figu
gure
gu
r 8B), pointing to
factor-1
targeted
arg
get
e ed effects
ts ooff ME
MEX
X on sspecific
peci
pe
cifi
ficc hy
fi
hypoxia-regulated
ypoxiaa-rregu
ulaated
d ssignaling
ignnali
ig
l ng
li
g ppathways.
athw
at
hwaays. T
hw
Treatment
reat
re
attme
ment
nt w
with
ithh an
it
equivalent
FEX
inhibitory
with
members
eq
equi
u va
ui
vale
l nt ddose
le
ose of F
ose
EX hhad
ad nno
o in
inhi
hibbito
hi
bito
ory eeffect
ffec
ectt on
ec
on tthe
hee hhypoxamirs
ypox
yp
oxxam
amirs
rss eexamined
xaamine
mineed wi
w
th oonly
nlly me
m
mber
mb
es
of the miR106b/25/93
miR10
06b
6b/2
/ 5/
/2
5/93
93 cluster
clu
l st
ster
err being
bei
eing
ei
ng moderately
mode
mo
dera
de
rate
ra
tely
te
ly affected
afffeccte
t d by FEX
FEX (Fi
(Fig
Figg 8A
Fi
8A).
).
Importantly, we observed that MEX treatment, but not FEX, resulted in the increase of lung
levels of miR-204 (Figure 8C), a microRNA enriched in distal pulmonary arterioles that is
transcriptionally suppressed by STAT3 but also inhibits the activation of STAT3 in a feed-forward
regulatory loop21. The proliferative and anti-apoptotic phenotype of PASMCs isolated from patients
with IPAH is inversely related to the level of miR-204 and delivery of exogenous miR-204 to the
lungs of animals with PH ameliorated established disease21. Therefore, we interpret these results as
an indication that MEX treatment, by suppressing STAT3 activation at the early stages of hypoxic
exposure, prevents the hypoxic induction of the pro-proliferative miR-17 superfamily in the lung
13
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DOI: 10.1161/CIRCULATIONAHA.112.114173
vasculature and blocks the STAT3-miR-204-STAT3 feed-forward loop in distal pulmonary vessels.
Discussion
This report, demonstrates that the protective functions of MSCs are mediated by secreted
microvesicles. Thus, our work provides an explanation for the paradox of the consistently
observed significant physiologic effect of MSC treatment despite very low retention of donor
cells in the lung. Secreted membrane microvesicles, of which exosomes represent the better
characterized subclass, have been recognized as important mediators of intercellular
communication, especially in the immune system. It has been proposed that such microvesicles,
called exosomes, can act as a vector for the transfer of genetic information (mRNA
(mRN
NA and
and
microRNAs) or the shuttling of effector proteins to recipient cells15, 16. Heat-shock protein 72
frrom
m tumor-derived
tumor
umor
or-d
-deriv
-d
iv
ved
ed exosomes mediates immunosuppressive
immunoosu
s ppressive function
funccti
t onn to myeloid-derived
myeloid-derived
from
suppressive
uppressive
ppp
cells
ceellls through
thro
rouugh
ugh activation
acti
ac
tivvati
ti
vation
on of
of STAT3
STA
AT319. T
Through
hro
oug
ughh pu
puta
putative
tati
tiivee ttransfer
raansfe
nsferr of m
microRNAs
icro
ic
ro
oRN
RNAs
As aand
ndd
mR
mRNAs,
RNA
NAs,
s, microvesicles
mic
icro
rovvessicl
siclees ssecreted
ecreete
ec
t d fr
from
om ttumor-initiating
um
mor
or-iini
niti
tiaatiing ce
cells
ell
llss po
positive
osi
sittive ffor
or tthe
or
he m
mesenchymal
e en
es
nch
chym
ym
mal
al m
marker
arke
ar
k r
ke
CD105 havee bbeen
eenn re
ee
repo
reported
port
po
r ed
rt
d tto
o co
confer
onf
n er ang
angiogenic
ngiiog
ng
ogen
en
nicc pphenotype
heno
he
n ty
type
pe tto
o no
norm
normal
rm
mal en
endo
endothelial
doth
do
t el
th
elia
iaal ce
cell
cells
l s37.
ll
Supporting our observations here, microvesicles released from MSCs have been recently
reported to improve recovery in animal models of experimentally-induced renal failure38 and
myocardial ischemia/reperfusion injury39, however, the underlying mechanisms mediating these
protective effects were not characterized.
Our results show that treatment with MSC-derived exosomes prevents the activation of
hypoxic signaling that underlies pulmonary inflammation and the development of PH in the
murine model. MCP-1 and HIMF/FIZZ1 are highly upregulated by hypoxia in the lung and both
factors are potent proinflammatory mediators. Moreover, both MCP-1 and HIMF/FIZZ1 have
14
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DOI: 10.1161/CIRCULATIONAHA.112.114173
been linked to the development of PH in murine models of disease40, 41 as well as in human PH42.
Egashira et. al. demonstrated that administration of exogenous recombinant MCP-1 resulted in
prominent medial wall thickening of pulmonary arterioles and MCP-1 receptor blockade
prevented monocyte recruitment as well as the subsequent vascular remodeling 41. HIMF/FIZZ1,
a marker of alternative activated macrophages43, is also induced by hypoxia in the respiratory
epithelium and plays a critical role in the development of HPH in a murine model and in
scleroderma-associated PH. 24, 44, 45. The knockdown of HIMF/FIZZ1 partially blocked increases
in pulmonary artery pressure, RVH, and vascular remodeling caused by chronic hypoxia. The
important role of HIMF in the development of PH was further confirmed through intrapulmonary
demo
demonstrate
monsstrrat
mo
atee here
here
he
r
gene transfer of HIMF/FIZZ1 which recapitulated the findings of HPH40. We de
that
hat MEX administration inhibited the hypoxic induction of both MCP-1 and HIMF/FIZZ1 in the
lung
ung and
and this
thi
hiss was
waas associated
as
d with prevention off HPH.
HPH.
HPH
Directl
Directly
tlyy related
r la
re
late
tedd to ooff th
thee an
ant
anti-inflammatory
ti-inf
ti-i
nfflam
mmatorry act
action
tion
tio
on ooff ME
MEX
EX tr
trea
treatment
eaatm
men
nt is
is tthe
he oobserved
bserrved
bs
ved
prevention
pathways.
Examining
lung
pr
prev
even
ev
enti
en
tion
on of
of hypoxia-activated
hy
ypo
oxi
xiaa-a
a-acti
tivaate
t d pro-proliferative
propr
o-pr
prrollif
ifer
erattiv
er
ivee pa
ath
hwaays
ys.. Ex
Exa
amin
amin
inin
ingg tota
ttotal
otaal lu
ung ttissue
is ue we
issu
found that, inn addition
add
d it
itio
ionn to MCP-1,
io
MCP
C -1
1, hypoxic
hyypooxi
x c levels
leve
le
v ls
ve
l of
of IL-6
I -66 were
IL
wer
eree also
als suppressed
sup
uppr
pres
pr
esse
es
s d by M
se
MEX
E
EX
administration. IL-6 is a proinflammatory cytokine known to activate STAT346 and, in a number
of studies, was associated with PH32, 47. It is thus possible that STAT3 may be a key mediator of
hypoxic, pro-inflammatory signaling leading to PH in the in vivo lung. Indeed, phosphorylation
of STAT3 at the 705 tyrosine residue is required for STAT3 dimerization and subsequent nuclear
translocation48, 49, and this was markedly increased in both lung and PAECs in response to
hypoxia but significantly suppressed by MEX treatment. Importantly, persistent ex vivo STAT3
activation, has been linked to the hyperproliferative and apoptosis-resistant phenotype observed
in PAECs27 and PASMCs28 from patients with IPAH. Mathew et. al. reported a marked
15
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DOI: 10.1161/CIRCULATIONAHA.112.114173
upregulation of STAT3 phosphorylation in the lungs of rats with monocrotaline-induced PH50.
STAT3 directly regulates the transcription of the miR-17~92 cluster of microRNAs in hPAECs,
resulting in decreased levels of BMPR2, a target of miR-1732 whose downregulation is
recognized as a hallmark of PH. We found that hypoxia induced select microRNAs of the miR17 superfamily in the lung and MEX effectively suppressed this induction whereas MEX did not
suppress other microRNAs involved in hypoxic signaling networks, including the hypoxamir
miR-210 which is induced by HIF36, pointing to selective, STAT3 targeted effects of MEX
action in the lung rather than global suppression of all hypoxamirs.
In addition to STAT3 being a central determinant of the hyperproliferative vascular cell
phenotype in patients with IPAH, the suppression off miR-204 (a distal pu
lmonar
aryy ar
aartery
teery sspecific
peci
pe
cific
pulmonary
microRNA) correlates with PH severity in human disease and rodent models of PH21. In this
tuddy, miR-204
miR
iR-2
-2004 w
-2
as suppressed by STAT3 and miR-204
as
miR
R-204 was show
ow
wn to
o iinhibit
nhibit the activation of
study,
was
shown
STAT3
STA
AT3 in a self-regulatory
sel
elff-reeguula
fl to
ory loop.
loo
oop.
p A
Although
l hough du
lt
duringg tthe
he aacute
cutee hhypoxic
cute
ypox
yp
oxxic ph
pha
phase
asee we
w ddid
id
d nnot
ot obs
observe
bser
bs
errvee
2
the
he decrease
decr
de
c ea
cr
ease
se iin
n miR-204
miR
R-204 le
R-2
leve
levels
els oobserved
bseerve
bs
erveed un
unde
under
derr ch
de
chronic
hro
oni
nicc hy
hyp
hypoxia
poxi
xiaa in tthe
xi
h m
he
mouse
ouse
ou
se21
we
we consider
connsid
id
der the
the
fact that ME
EX tr
trea
eaatm
tmen
entt in
en
ncr
crea
e se
sess th
the ba
bbasal
sall le
sa
eve
v l of m
iR
R-2
204 as a sstrong
t on
tr
ongg in
indi
d ca
di
cati
tion
ti
on tthat
h t MEX
ha
MEX
treatment
increases
level
miR-204
indication
treatment is shifting the balance of the STAT3-miR-204 loop to an anti-proliferative state.
A schema of a hypothesis synthesizing the above results with previous work from our
group and the work of others is shown in Supplemental Figure 5. We have previously shown
that hypoxia shifts the Th1/Th2 balance of immunomodulators in the lung, resulting in
alternative activated alveolar macrophages (AA-AMĭ) and this is inhibited by HO-1
overexpression2. Hypoxia also induces the expression of HIMF in the lung epithelium24 and
HIMF mitogenic action on the vasculature requires Th2 cytokines, such as IL-444 to result in PH.
Consequences of the shift towards proliferation include the hypoxic activation of STAT3
16
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signaling and the upregulation of the miR-17 family of microRNAs. Treatment with MEX
interferes with an early hypoxic signal in the lung, suppressing inflammation, HIMF
transcriptional upregulation, and alternative macrophage activation. It addition, MEX treatment
may directly suppress STAT3 activation in lung vascular cells and also upregulate miR-204
levels, thus breaking the STAT3-miR-204-STAT3 feed-forward loop and shifting the balance to
an anti-proliferative state.
The ability to secrete microparticles that contain not only proteins but RNA or miRNA
species which can modulate the expression of multiple genes make these packaging vesicles an
attractive and quite plausible means for MSCs to regulate multiple pathways and produce a
robust
endocytic
obust therapeutic effect in vivo. Indeed, exosomes are lipid vesicles of endocyti
ticc or
oorigin
ig
gin released
rel
elea
e se
ea
s d
by many cell types including vascular cells, dendritic cells, and mast cells13 that function to
medi
me
diat
di
atee intercellular
at
inte
in
terrcelllu
te
lullar
la communication through the
th
he ex
xchange of prot
oteinn an
ot
andd RNA moieties. Of
mediate
exchange
protein
possible
poss
ssib
si le physiologic
physiiollog
ogicc relevance
rel
elev
evan
ev
ance
an
ce is
is the
the ddifferential
ifffere
ff ential ddistribution
istrib
ib
butio
utio
on of ttetraspanins
ettras
trasppannins
nss C
CD63
D663 (a
(abu
(abundant
b nd
bu
ndan
an
nt iin
n
mouse
mo
ous
usee MEX)
ME
EX) and
and CD81
CD81 (abundant
(abu
(a
bu
und
dan
antt in
in mouse
mou
ouse
se FEX).
FEX
EX).. Although
Alth
Alth
thou
ough
ou
gh
h this
thiss differential
d ffferren
di
enti
tiaal di
ti
dis
distribution
stribu
str
ribu
utioon
on is
is not
no
apparent betwe
between
ween
we
e hhuman
en
uman
um
an M
MEX
E aand
EX
n hhuman
nd
um
man F
FEX
EX ((results
resu
re
s ltts not
su
not shown),
shhow
own)
n),, th
n)
thes
these
esee ar
es
aaree mo
mole
molecules
lecu
le
c les that
cu
may play a role in target cell specificity of exosomes and could participate in signaling pathways.
Full molecular characterization of exosomal preparations produced by mouse bone marrow
MSCs and human umbilical cord stroma MSCs is the focus of ongoing work. Exosomes isolated
from a mast-cell line or from primary bone marrow-derived mast cells were reported to contain
mRNAs and microRNAs that were transferable to other mast cells, and, in the case of mRNAs,
to be translated into new proteins13. The authors identified different miRNAs within exosomes
and, in a more recent study, pre- and mature miRNAs, but not larger species, were identified
within MSC-derived exosomes51. Given the robust, long-lasting anti-inflammatory and
17
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DOI: 10.1161/CIRCULATIONAHA.112.114173
cytoprotective effects of MEX demonstrated for the first time in the present study, it is
reasonable to postulate that one or more miRNA species that are unique to or highly enriched
within MEX, serve as master regulator(s) of several genes and pathways underlying the
development of PH.
In summary, in this study we isolated, identified, and characterized exosomes from
mouse and human MSC-CM and demonstrated a robust biologic effect that is unique to MEX
versus exosomes derived from other cells, such as fibroblasts. Importantly, we demonstrate for
the first time that MEX are the major paracrine anti-inflammatory and therapeutic mediators of
MSC action on the lung, acting, at least in part, through inhibition of hypoxic STAT3 signaling.
protein,
lipid,
Further work is required to identify the critical components of MEX, be they pro
otein
tein
n, li
lipi
pid,
pi
d, oorr
nucleic acid species. Although the applicability of our findings to a human disease model need
to
verified,
the
makes
o bbee ve
rifi
ri
ifi
fied
ed, th
ed
he ef
eefficacy
ficacy of this treatment in ppreventing
revventing PH mak
re
kes
e tthese
hesse
he
se microvesicles an
attractive
candidate
PH
diseases
at
ttrractive
ac
candi
dida
d te for
for exploring
expplo
ori
ring
ng models
mod
odel
elss off therapeutic
thheraapeutiic interventions
intterven
in
nti
tion
ons in
i P
H and
and othe
oother
the
herr di
dis
seassess
seas
with
date.
wi
ith no
no definitive
defi
de
fini
fi
niti
tivve ttherapy
herrappy
he
py too da
date
te.
te
Acknowledgements:
expertise,
Sarah
Ackn
Ac
know
owle
ledg
dgem
emen
ents
ts:: Th
Thee authors
auth
au
thor
orss would
woul
wo
uldd like
like to
to thank
than
th
ankk Xianlan
Xian
Xi
anla
lann Liu
Liu for
for technical
tech
te
chni
nica
call ex
expe
pert
rtis
isee S
arah
ar
ah
h
Gately for assistance in preparing the manuscript, and Dr. Georg Hansmann for critical review of
the manuscript.
Funding Sources: This work was supported by NIH RO1 HL055454 and NIH RO1 HL085446
(SK & SAM).
Conflict of Interest Disclosures: None.
18
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DOI: 10.1161/CIRCULATIONAHA.112.114173
References:
1. Minamino T, Christou H, Hsieh CM, Liu Y, Dhawan V, Abraham NG, Perrella MA, Mitsialis
SA, Kourembanas S. Targeted expression of heme oxygenase-1 prevents the pulmonary
inflammatory and vascular responses to hypoxia. Proc Natl Acad Sci U S A. 2001;98:8798-8803.
2. Vergadi E, Chang MS, Lee C, Liang OD, Liu X, Fernandez-Gonzalez A, Mitsialis SA,
Kourembanas S. Early Macrophage Recruitment and Alternative Activation Are Critical for the
Later Development of Hypoxia-Induced Pulmonary Hypertension. Circulation. 2011;123:19861995.
3. Weiss DJ, Bertoncello I, Borok Z, Kim C, Panoskaltsis-Mortari A, Reynolds S, Rojas M,
Stripp B, Warburton D, Prockop DJ. Stem cells and cell therapies in lung biology and lung
diseases. Proc Am Thorac Soc. 2011;8:223-272.
4. Rojas M, Xu J, Woods CR, Mora AL, Spears W, Roman J, Brigham KL. Bone marrowderived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol.
2005;33:145-152.
DG.
5. Ortiz LA, Gambelli F, McBride C, Gaupp D, Baddoo M, Kaminski N, Phinney
ey D
G.
Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and
2003;100:8407-8411.
ameliorates its fibrotic effects. Proc Natl Acad SSci
ci U S A. 2003;100:84077 8411.
6. Xu
Xu J,
J Woods
Wooods
d CR,
CR,
R Mora
Mor
o a AL,
A , Joodi
AL
Jood
Jo
o i R,, Brigham
od
Bri
righ
g am KL,
gh
KL, Iyer
Iy
yer S,
S, Rojas
Rooja
jass M. Prevention
Pre
r ve
vent
ntio
nt
ionn of endotoxinio
end
ndot
otox
ot
oxin
ox
i induced
systemic
marrow-derived
mesenchymal
mice.
ndu
uce
c d system
mic rresponse
esspo
ponnse
nse by bbone
on
ne ma
mar
rrow-d
-deerivvedd mes
m
esen
senchym
hymal
mal st
stem
em
m ccells
ells
el
ls iin
n mi
m
icce.
ce. Am J
Physiol
Cell
P
Phys
hys
ysiol Lung
ng
g Ce
el Mol
ell
Mo
ol Physiol.
Phys
yssio
ol.
l 20
22007;293:L131-141.
07;2
07
;2
293:L
L1331-11411.
Kourembanas
7. Aslam
Asl
slam
a M,
M, Baveja
Ba ja R,
R, Liang
Lian
Li
a g OD,
OD Fernandez-Gonzalez
Fernan
ande
dezz Go
Gonzaale
lezz A, Lee
Lee C,
C, Mitsialis
Mits
Mi
tsiali
liss SA,
SA, Ko
K
urem
ur
emba
bana
nass S.
S
chronic
Bone marrow
w stromal
stro
st
rooma
m l cells
ceellss attenuate
atte
at
tenu
nuat
nu
atte lung
lu
ung injury
inj
n ur
uryy in a murine
mur
urin
inee model
in
mode
mo
d l of nneonatal
eona
eo
n taal ch
na
chro
roni
ro
n c lung
ni
disease.
dise
di
seas
asee Am J Respir
Res
espi
pirr Crit
Crit Care
Car
aree Med.
Medd 2009;180:1122-1130.
Me
2009
20
09;1
;180
80:1
:112
1222-11
1130
30
8. van Haaften T, Byrne R, Bonnet S, Rochefort GY, Akabutu J, Bouchentouf M, Rey-Parra GJ,
Galipeau J, Haromy A, Eaton F, Chen M, Hashimoto K, Abley D, Korbutt G, Archer SL,
Thebaud B. Airway delivery of mesenchymal stem cells prevents arrested alveolar growth in
neonatal lung injury in rats. Am J Respir Crit Care Med. 2009;180:1131-1142.
9. Baber SR, Deng W, Master RG, Bunnell BA, Taylor BK, Murthy SN, Hyman AL, Kadowitz
PJ. Intratracheal mesenchymal stem cell administration attenuates monocrotaline-induced
pulmonary hypertension and endothelial dysfunction. Am J Physiol Heart Circ Physiol.
2007;292:H1120-1128.
10. Liang OD, Mitsialis SA, Chang MS, Vergadi E, Lee C, Aslam M, Fernandez-Gonzalez A,
Liu X, Baveja R, Kourembanas S. Mesenchymal stromal cells expressing heme oxygenase-1
reverse pulmonary hypertension. Stem Cells. 2011;29:99-107.
11. Hansmann G, Fernandez-Gonzalez A, Aslam M, Vitali SH, Martin T, Mitsialis SA,
19
Downloaded from http://circ.ahajournals.org/ by guest on July 24, 2016
DOI: 10.1161/CIRCULATIONAHA.112.114173
Kourembanas S. Mesenchymal stem cell-mediated reversal of bronchopulmonary dysplasia and
associated pulmonary hypertension. Pulm Circ. 2012;2:170-81.
12. Lee JW, Fang X, Krasnodembskaya A, Howard JP, Matthay MA. Concise review:
Mesenchymal stem cells for acute lung injury: role of paracrine soluble factors. Stem Cells.
2011;29:913-919.
13. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated
transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
Nat Cell Biol. 2007;9:654-659.
14. Aoki N, Jin-no S, Nakagawa Y, Asai N, Arakawa E, Tamura N, Tamura T, Matsuda T.
Identification and characterization of microvesicles secreted by 3T3-L1 adipocytes: redox- and
hormone-dependent induction of milk fat globule-epidermal growth factor 8-associated
microvesicles. Endocrinology. 2007;148:3850-3862.
15. Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat
Rev Immunol. 2002;2:569-579.
cancer-associated
16. Taylor DD, Gercel-Taylor C. Exosomes/microvesicles: mediators of cancer-as
asso
soci
so
ciat
ated
at
ed
immunosuppressive
mmunosuppressive microenvironments. Semin Immunopathol. 2011;33:441-454.
17.
17
7. Segura
Seggura
Se
guraa E,
E, Guerin
Guuer
erin
i C, Hogg N, Amigorena S, Thery
Thery C. CD8+ dendritic
The
d nd
de
dri
riti
ticc cells use LFA-1 to
ti
capture
2007;179:1489-1496.
ca
aptture MHC-peptide
MH
HC-pe
pept
ptid
pt
idee complexes
c mp
co
mple
lexe
xees from
f om exosomes
fr
exo
xoso
some
mes inn vivo.
viv
vo. J Im
IImmunol.
munol.
mun
nol. 20
007
0 ;1
;179
79:1
79
:148
:1
4899-14
14
496
6.
18.
derived
from
M.. Bo
Bovis
BCG
macrophages
18
8. Giri
Giri PK,, Schorey
Sch
horrey JS.
JS. Exosomes
Exos
xosome
som s de
eriivedd fr
rom M
Bovi
viss B
vi
CG
G iinfected
nfeccted m
acr phhages activate
acro
activ
vate
antigen-specific
CD4+
cells
vitro
and
vivo.
2008;3:e2461.
an
nti
tige
genge
n sp
nspec
eciific
ec
ific C
D4+ and
D4+
and CD
CD8+
8+ T ce
ell
llss in vit
itro
it
r an
ro
nd iin
n vi
viv
vo. PL
vo.
PLoS
oSS One.
One.
ne. 20
008
08;3
; :ee246
;3
2461.
19. Chalminn F, Ladoire
Lad
adoi
o re
oi
r S, Mignot
Mign
Mi
gn
not G,
G, Vincent
Vinc
Vi
ncen
nc
entt J,
en
J Bruchard
Bru
ruch
c ar
ch
ardd M,
M Remy-Martin
Rem
e yy-Ma
Mart
Ma
rtin
rt
in
n JP,
JP,
P Boireau
Boi
oire
reau
re
a W,
Rouleau
A, S
Simon
B, La
Lanneau
D, D
Dee Th
Thonel
A, Mu
Multhoff
G, Ha
Hamman
A, Ma
Martin
F, Ch
Chauffert
B,
Roul
Ro
ulea
eauu A
imon
im
on B
Lann
nnea
eauu D
Thon
onel
el A
Mult
ltho
hoff
ff G
Hamm
mman
an A
Mart
rtin
in F
Chau
auff
ffer
ertt B
Solary E, Zitvogel L, Garrido C, Ryffel B, Borg C, Apetoh L, Rebe C, Ghiringhelli F.
Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent
immunosuppressive function of mouse and human myeloid-derived suppressor cells. J Clin
Invest. 2010;120:457-471.
20. Ratajczak J, Miekus K, Kucia M, Zhang J, Reca R, Dvorak P, Ratajczak MZ. Embryonic
stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal
transfer of mRNA and protein delivery. Leukemia. 2006;20:847-856.
21. Courboulin A, Paulin R, Giguere NJ, Saksouk N, Perreault T, Meloche J, Paquet ER, Biardel
S, Provencher S, Cote J, Simard MJ, Bonnet S. Role for miR-204 in human pulmonary arterial
hypertension. J Exp Med. 2011;208:535-548.
22. Vitali SH, Mitsialis SA, Liang OD, Liu X, Fernandez-Gonzalez A, Christou H, Wu X,
McGowan FX, Kourembanas S. Divergent cardiopulmonary actions of heme oxygenase
enzymatic products in chronic hypoxia. PLoS One. 2009;4:e5978.
20
Downloaded from http://circ.ahajournals.org/ by guest on July 24, 2016
DOI: 10.1161/CIRCULATIONAHA.112.114173
23. Zampetaki A, Minamino T, Mitsialis SA, Kourembanas S. Effect of heme oxygenase-1
overexpression in two models of lung inflammation. Exp Biol Med. 2003;228:442-446.
24. Teng X, Li D, Champion HC, Johns RA. FIZZ1/RELMalpha, a novel hypoxia-induced
mitogenic factor in lung with vasoconstrictive and angiogenic properties. Circ Res.
2003;92:1065-1067.
25. Hokuto I, Ikegami M, Yoshida M, Takeda K, Akira S, Perl AK, Hull WM, Wert SE, Whitsett
JA. Stat-3 is required for pulmonary homeostasis during hyperoxia. J Clin Invest. 2004;113:2837.
26. Quinton LJ, Jones MR, Robson BE, Simms BT, Whitsett JA, Mizgerd JP. Alveolar epithelial
STAT3, IL-6 family cytokines, and host defense during Escherichia coli pneumonia. Am J Respir
Cell Mol Biol. 2008;38:699-706.
27. Masri FA, Xu W, Comhair SA, Asosingh K, Koo M, Vasanji A, Drazba J, Anand-Apte B,
pulmonary
Erzurum SC. Hyperproliferative apoptosis-resistant endothelial cells in idiopathicc pu
pulm
lmon
onar
aryy
arterial hypertension. Am J Physiol Lung Cell Mol Physiol. 2007;293:L548-554..
28. Paulin R, Courboulin A, Meloche J, Mainguy V, Dumas de la Roque E, Saksouk N, Cote J,
Provencher S,
S, Sussman
Suussman MA, Bonnet S. Signal transducers and activators of transcription-3/pim1
axis
ax
xiss plays
pla
lays
yss a critical
cri
r tiica
call role in the pathogenesis of human
hum
hu
man pulmonaryy arterial
a teeri
ar
rial
al hypertension.
Circulation.
Circ
Ci
cul
ulation. 2011;123:1205-1215.
2011
2011
1;1
; 23
23:1
:120
:1
20
055 12
1215
15..
15
29.
Helwig
29
9. Mitchell
Mitchelll KE,
KE,, Weiss
Weisss ML,
ML
L, Mitchell
Mittch
chel
e l BM,
el
BM Martin
M rtinn P,
Ma
P, Davis
Dav
avis
is D,
D, Morales
Mo s L,
L, H
elwi
w g B,
B,
Beerenstrauch
M,, A
Abou-Easa
K,, Hi
Hildreth
T,, Tr
Troyer
D,, Me
Medicetty
Matrix
cells
Wharton's
Beer
Be
e en
er
enst
stra
rauc
uchh M
uc
boou-Ea
Easa
saa K
Hild
ldrreth
reth T
Troy
oyyerr D
Med
dice
dice
cett
ttty S.
S. M
attri
rixx ce
ell
llss fr
from
om W
harrtoon's
ha
on
jelly
glia.
Cells.
2003;21:50-60.
ellly fo
form
m nneurons
eu
uro
ronss aand
nd gli
lia.
a St
Stem
em C
ells. 20
2003
03;2
;21:
1:550-6
60.
0
30. Montecalvo
A, Larregina
AT, Shufesky
WJ,
DB, Su
Sullivan
ML,
Karlsson
JM, Ba
CJ,
30
Mont
Mo
ntec
ecal
alvo
vo A
Larr
La
rreg
egin
inaa AT
Shuf
Sh
ufes
esky
ky W
J Stolz
Stol
olzz DB
Sull
lliv
ivan
an M
L K
arls
ar
lsso
sonn JM
Baty
ty C
J
Gibson GA, Erdos G, Wang Z, Milosevic J, Tkacheva OA, Divito SJ, Jordan R, Lyons-Weiler J,
Watkins SC, Morelli AE. Mechanism of transfer of functional microRNAs between mouse
dendritic cells via exosomes. Blood. 2012;119:756-766.
31. Mittelbrunn M, Gutierrez-Vazquez C, Villarroya-Beltri C, Gonzalez S, Sanchez-Cabo F,
Gonzalez MA, Bernad A, Sanchez-Madrid F. Unidirectional transfer of microRNA-loaded
exosomes from T cells to antigen-presenting cells. Nat Commun. 2011;2:282.
32. Brock M, Trenkmann M, Gay RE, Michel BA, Gay S, Fischler M, Ulrich S, Speich R, Huber
LC. Interleukin-6 modulates the expression of the bone morphogenic protein receptor type II
through a novel STAT3-microRNA cluster 17/92 pathway. Circ Res. 2009;104:1184-1191.
33. Cloonan N, Brown MK, Steptoe AL, Wani S, Chan WL, Forrest AR, Kolle G, Gabrielli B,
Grimmond SM. The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle
transition. Genome Biol. 2008;9:R127.
21
Downloaded from http://circ.ahajournals.org/ by guest on July 24, 2016
DOI: 10.1161/CIRCULATIONAHA.112.114173
34. Rane S, He M, Sayed D, Vashistha H, Malhotra A, Sadoshima J, Vatner DE, Vatner SF,
Abdellatif M. Downregulation of miR-199a derepresses hypoxia-inducible factor-1alpha and
Sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes. Circ Res.
2009;104:879-886.
35. Watanabe T, Sato T, Amano T, Kawamura Y, Kawamura N, Kawaguchi H, Yamashita N,
Kurihara H, Nakaoka T. Dnm3os, a non-coding RNA, is required for normal growth and skeletal
development in mice. Dev Dyn. 2008;237:3738-3748.
36. Chan SY, Loscalzo J. MicroRNA-210: A unique and pleiotropic hypoxamir. Cell Cycle.
2010;9:1072-1083.
37. Grange C, Tapparo M, Collino F, Vitillo L, Damasco C, Deregibus MC, Tetta C, Bussolati
B, Camussi G. Microvesicles released from human renal cancer stem cells stimulate
angiogenesis and formation of lung premetastatic niche. Cancer Res. 2011;71:5346-5356.
38. Bruno S, Grange C, Deregibus MC, Calogero RA, Saviozzi S, Collino F, Morando L, Busca
microvesicles
A, Falda M, Bussolati B, Tetta C, Camussi G. Mesenchymal stem cell-derived mic
icro
rove
ro
vesi
sicl
cles
es
protect against acute tubular injury. J Am Soc Nephrol. 2009;20:1053-1067.
CN,
39. Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS, Salto-Tellez M, Timmers L, Lee CN
El Oakley RM, Pasterkamp G, de Kleijn DP, Lim SK. Exosome secreted by MSC reduces
myocardial
Cell
Res.
2010;4:214-222.
my
yocar
ocar
ardi
dial
di
al iischemia/reperfusion
sche
heemia/
mia reperfusion injury. Stem C
el R
ell
es. 2010;4:214
1 -222
14
22..
22
Angelini
DJ,
Su
Q,, Y
Yamaji-Kegan
K,, Fa
Fan
Skinner
Champion
HC,
MT,
440.
0. A
ngelini D
J, S
J,
uQ
amaj
am
ajii-Ke
aj
i-Keegan
gan K
an C, S
an
kinn
ner
er JJT,
T C
T,
ham
ha
mpio
mpio
i n HC
C, Crow
Crow
wM
T, JJohns
ohns
oh
ns
Hypoxia-induced
mitogenic
factor
induces
vascular
and
RA. Hy
RA.
H
poxiiaa-indu
ducedd m
du
ito
ogen
genicc fa
act
c or ((HIMF/FIZZ1/RELMalpha)
HIMF
MF/FIIZZ1//RE
MF
RELM
LMalppha
LM
pha) in
nduuce
uces tthe
he va
ascula
ascu
larr an
nd
hemodynamic
changes
Physiol
Lung
Mol
Physiol.
hemo
he
mody
mo
dyna
dy
nami
micc ch
mi
han
ngees of
of ppulmonary
ulmo
ul
mo
ona
narry
ry hhypertension.
ypper
erttens
nsio
ns
ion.
n. Am
mJP
hysi
hy
s ol
si
ol L
u g Cell
un
Celll M
o P
ol
hy ioll.
hysi
2009;296:L582-593.
2009
09;2
;296
96:L
:L58
822-59
93.
41. Eg
Egashira
K, Ko
Koyanagi
M, Ki
Kitamoto
S, Ni W
W, K
Kataoka
C, Mo
Morishita
R, K
Kaneda
Y, A
Akiyama
41
Egas
ashi
hira
ra K
Koya
yana
nagi
gi M
Kita
tamo
moto
to S
atao
at
aoka
ka C
Mori
rish
shit
itaa R
aned
an
edaa Y
kiya
ki
yama
ma
C, Nishida KI, Sueishi K, Takeshita A. Anti-monocyte chemoattractant protein-1 gene therapy
inhibits vascular remodeling in rats: blockade of MCP-1 activity after intramuscular transfer of a
mutant gene inhibits vascular remodeling induced by chronic blockade of NO synthesis. FASEB
J. 2000;14:1974-1978.
42. Itoh T, Nagaya N, Ishibashi-Ueda H, Kyotani S, Oya H, Sakamaki F, Kimura H, Nakanishi
N. Increased plasma monocyte chemoattractant protein-1 level in idiopathic pulmonary arterial
hypertension. Respirology. 2006;11:158-163.
43. Raes G, De Baetselier P, Noel W, Beschin A, Brombacher F, Hassanzadeh Gh G.
Differential expression of FIZZ1 and Ym1 in alternatively versus classically activated
macrophages. J Leukoc Biol. 2002;71:597-602.
44. Yamaji-Kegan K, Su Q, Angelini DJ, Myers AC, Cheadle C, Johns RA. Hypoxia-induced
mitogenic factor (HIMF/FIZZ1/RELMalpha) increases lung inflammation and activates
pulmonary microvascular endothelial cells via an IL-4-dependent mechanism. J Immunol.
22
Downloaded from http://circ.ahajournals.org/ by guest on July 24, 2016
DOI: 10.1161/CIRCULATIONAHA.112.114173
2010;185:5539-5548.
45. Angelini DJ, Su Q, Yamaji-Kegan K, Fan C, Teng X, Hassoun PM, Yang SC, Champion
HC, Tuder RM, Johns RA. Resistin-like molecule-beta in scleroderma-associated pulmonary
hypertension. Am J Respir Cell Mol Biol. 2009;41:553-561.
46. Bromberg J, Darnell JE, Jr. The role of STATs in transcriptional control and their impact on
cellular function. Oncogene. 2000;19:2468-2473.
47. Steiner MK, Syrkina OL, Kolliputi N, Mark EJ, Hales CA, Waxman AB. Interleukin-6
overexpression induces pulmonary hypertension. Circ Res. 2009;104:236-244.
48. Yahata Y, Shirakata Y, Tokumaru S, Yamasaki K, Sayama K, Hanakawa Y, Detmar M,
Hashimoto K. Nuclear translocation of phosphorylated STAT3 is essential for vascular
endothelial growth factor-induced human dermal microvascular endothelial cell migration and
tube formation. J Biol Chem. 2003;278:40026-40031.
49. Hirano T, Ishihara K, Hibi M. Roles of STAT3 in mediating the cell growth, differentiation
difffe
f re
rent
ntia
iati
tion
on
and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene.
Onco
ogene
n .
2000;19:2548-2556.
50. Mathew R, Huang J, Shah M, Patel K, Gewitz M, Sehgal PB. Disrup
Disruption
uption of endothelial-cell
caveolin-1alpha/raft
development
monocrotaline-induced
caave
veol
olin
ol
in-1
-1al
1al
alph
p a//ra
raft
ft scaffolding during developm
men
nt of monocro
ota
taline
ne-i
-iinduced pulmonary
hypertension.
hyypeerttensiion
n. Circulation.
Circ
Ci
rcul
rc
ulat
atio
ion.
io
n. 2004;110:1499-1506.
2 04
20
4;1
;110
10:1
:149
4999-15
1506
06.
51.
Chen
Lai
RC,
Lee
MM,
Choo
AB,
CN,
Lim
Mesenchymal
51. C
hen TS,
S, La
ai R
C, L
ee M
M, C
M,
hooo A
B, Lee
Le CN
N, Li
im SK
SK.. Me
esenchhym
mal sstem
mal
tem ccell
elll ssecretes
eccretees
es
microparticles
pre-microRNAs.
Acids
miicr
c op
opar
arti
ticl
clees
cl
es eenriched
nriche
nri
icheed in pre
re-m
re
mic
icro
roRN
ro
RN
NAs
As.. Nucleic
Nucl
Nu
c eicc A
cl
cid
ds Re
Res.
s. 2010;38:215-224.
2010
2010
10;3
;388:21
2 5-22
21
5-22
224.
4.
Figure
Figu
Fi
gure
re Legends:
Leg
egen
ends
ds::
Figure 1. MSC-CM but not MLF-CM suppress hypoxia-induced pulmonary influx of
macrophages. (A) Macrophage numbers in the BALF of hypoxic mice and littermate normoxic
controls. Animals received either serum-free culture media (vehicle), MSC-CM (5 μg protein) or
MLF-CM (5 μg protein) prior to hypoxic exposure for 48 hours. Each dot represents the total
number of Kimura stain-positive cells in BALF from individual animals. Horizontal lines
represent the group mean and vertical brackets the standard deviation. (B) BALF levels of MCP1 and HIMF. Cell-free BALF specimens from all animals in each treated group were pooled and
23
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DOI: 10.1161/CIRCULATIONAHA.112.114173
an amount corresponding to one animal equivalent was subjected to western blot analysis. IgA
levels were assessed as a control for equal loading. n > 6 for all groups. ¶, p<0.001 vs. untreated
normoxic controls; §, p<0.001 vs. PBS-treated hypoxic controls (One-way ANOVA with
Tukey-Kramer post-test).
Figure 2. (A) Size fractionation of concentrated conditioned media by Sephacryl-400
chromatography. The black trace represents elution of protein (A280) and the gray trace
represents the elution profile of 50 nm diameter polystyrene nanospheres (A220). The fractions of
conditioned media in the retention volume corresponding to the 50 nm diameter range were
pooled and termed Fraction I, and fractions representing moieties of smaller sizee we
were
eree ppooled
oole
oo
ledd as
le
a
Fraction II. (B) Negative staining electron microscopy at 30,000X magnification revealed
heterogeneous
he
eteero
roggene
gene
neou
ous microvesicles
ou
miccro
mi
crovesicles specifically in Frac
Fraction
cti
t on I. (C) Fracti
Fraction
ion
o I ppreparations
repa
re
p rations from either
MEX
ME
X or FEX were
weree enriched
en
nri
rich
ched
ch
ed in
in 30-100
300-1
-100
000 nm
nm diameter
diaametter m
microvesicles
iccro
rovvesi
sicl
cles
es eexhibiting
xh
hib
ibiitin
in
ng ty
typi
typical
piica
call ex
exosome
xos
osom
om
me
morphology
(D)
Preparations
either
MSCs
MLFs
mo
orp
rpho
holo
ho
logy
gy (arrows).
(ar
arrrows).
ws).. (D
D) P
r pa
re
para
rati
ra
tion
ti
on
ns of eexosomes
xo
oso
s me
mes pr
pproduced
oduuced
od
ed bby
y ei
eith
th
her M
SCss orr M
SC
LFs ar
LFs
aree
associated with
witth typical
typi
ty
pica
pi
call exosomal
ca
e os
ex
osom
omal
om
al markers
mar
a ke
kers
rss as
as well
welll as Dicer.
Diccer
er.. The
The relative
reela
l ti
tive
ve levels
lev
evel
ells of tthese
hese markerss
he
depends on the cellular origin of the microvesicles. Density for each band relative to Alix was
measured and relative abundance in MEX compared to FEX for each marker is shown on right.
(E) Macrophage numbers in the BALF of animals exposed to hypoxia for 48 hours and treated
with either vehicle (PBS), MEX, FEX, or the exosome-depleted fraction of MSC-CM (ExDCM). Normoxic animals were used as a control. ¶, p<0.001 vs. normoxic control animals; §,
p<0.001 vs. PBS-treated hypoxic animals (One-way ANOVA with Tukey-Kramer post-test). (F)
Western blot analysis of MCP-1 and HIMF levels in BALF of hypoxic animals treated with
isolated exosomes and controls.
24
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DOI: 10.1161/CIRCULATIONAHA.112.114173
Figure 3. Repeated administration of a low MEX dose (x2) can suppress pulmonary influx of
macrophages and ameliorate HPH. (A) Animals were injected with one dose of PBS (left panel,
n=14) or 0.1 μg MEX (Middle panel, n=31) through the jugular vein prior to hypoxic exposure
and alveolar macrophages were measured in BALF at sacrifice on the days indicated on the X
axis. Animals (Right panel, n=15) received 0.1 μg MEX prior to exposure to hypoxia and 0.1 μg
MEX again at day 4 of hypoxia and alveolar macrophages were measured in BALF at sacrifice
on the days indicated on the X axis. Each dot represents an individual animal. Macrophage
numbers in BALF from normoxic control animals (open circles) are replicated in all three panels
for direct comparison. ¶, p<0.001 vs. normoxic control group (One-way ANOVA with TukeyBALF
Kramer post-test). (B) Representative Western blot analysis of cytokine levels in
nB
ALF
AL
F of
animals treated with a single low dose MEX. BALF from 6 animals in each group was pooled
for
times.
fo
or immunoblot
imm
im
muno
muno
nobl
b ot aanalysis
bl
na
nalysis
and repeated at least 3 ti
tim
mes. (C) RVSP
P and
andd (D)
(D) Fulton’s Index
determined
de erm
dete
r ined after
aft
fter
er 3 weeks
weeks of
of hypoxic
hypo
hy
poxi
xicc exposure.
xi
exposuuree. The
he Fulton’s
Fuult
ulton’
ton’’s Index
Inde
In
dexx (RV/[LV+S])
(RV/
(R
V [L
V/
[LV+
V+S]
V+
S])) iiss a
measurement
ratio.
RV,
meeas
a ur
urem
emen
entt of right
en
rig
ght ventricular
ventr
entriccul
ulaar hypertrophy
hype
hy
pert
r ro
rt
roph
phyy expressed
ph
e preesssed as a weight
ex
weighht ra
atioo. R
V, rright
ight
ig
ht vventricle;
entr
entr
t ic
iclle;;
LV, left ventr
ventricle;
Animals
with
MEX(x2),
trric
icle
l ; S, sseptum.
le
ep
ptu
um. ME
MEX
X (x
((x1):
1):: An
1)
Anim
imal
im
alss we
al
were
re iinjected
njec
nj
ecte
ec
teed on
once
ce w
itth ME
MEX.
X M
X.
E (x2),
EX
FEX(x2): Animals were injected twice with equivalent amounts of MEX or FEX. Black dots
represent values for individual animals, horizontal lines represent the group mean and vertical
brackets the standard deviation. ¶, p<0.001 vs. normoxic control group; ‡, p<0.01 vs. normoxic
control group; §, p<0.001 vs. PBS-injected hypoxic control group (One-way ANOVA with
Tukey-Kramer post-test).
Figure 4. Repeated administration of low MEX dose (x2) inhibits lung vascular remodeling.
Representative lung sections are shown from animals exposed to the same conditions as
described in the legend of Figure 3 and stained for Į-SMA to evaluate medial vessel wall
25
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DOI: 10.1161/CIRCULATIONAHA.112.114173
thickness. (A) 100X magnification and (B) 400X.
Figure 5. A single high dose MEX efficiently suppresses the entire period of inflammatory
responses to early hypoxia. (A) Macrophage numbers in the BALF of animals injected with a
high dose (10 Pg) MEX prior to hypoxic exposure for 2 and 7 days. Dots represent values for
individual animals. Black dots represent values for MEX-treated group. White and gray dots
represent values for normoxic group (NRX) and PBS-treated groups, respectively, which are
adopted from Fig 3A. Horizontal lines represent the group mean and vertical brackets the
standard deviation. ¶, p<0.001 vs. normoxic control group (One-way ANOVA with TukeyBALF
Kramer post-test). (B) Representative Western blot analysis of FIZZ-1/HIMF llevels
evel
ev
elss in B
el
ALF
AL
F oof
animals treated with a single high dose MEX and FEX (10 μg) prior to hypoxic exposure for 7
days.
days
ys..
Figure
single
dose
RVSP
measurements
Fi
igu
ure 6. A si
sing
glee high
hig
gh do
ose MEX
MEX treatment
tre
reat
atm
mennt prevents
prevvent
ventss HPH.
HPH
HP
H. (A)) R
VSP
Pm
eassure
r mennts
nts aand
nd ((B)
B)
Fulton’s
normoxic
animals
injected
with
either
PBS
μg
MEX
Fult
lton
on’s
’ IIndex
ndex
nd
x of no
norm
rmox
xic
i aanimals,
nima
ni
mals
l , andd an
anim
imal
als inje
j ct
je
cted
ed w
ithh ei
it
ith
ther
er P
BS
S oorr 10 μ
gM
EX
X pr
pprior
iorr
too a three
animals,
three weekk hypoxic
hypoxic
i exposure.
exposure Black
Black
k dots
dots
t representt values
vallues for
for iindividual
ndi
divid
iduall ani
imals
l hhorizontal
oriizonttall
lines represent the group mean and vertical brackets the standard deviation. ¶, p<0.001 vs.
normoxic control group; §, p<0.001 vs. PBS-treated hypoxic control group (One-way ANOVA
with Tukey-Kramer post-test). (C) Histology of paraffin-embedded lung sections stained for
DSMA at 400X magnification, depicting representative pulmonary arterioles. (D) Assessment of
medial wall thickness of small pulmonary arterioles (20-40 μm diameter) in the three
experimental groups (sections from four animals per group; n>40 vessels measured per animal).
¶, p<0.001 vs. normoxic control group; §, p<0.001 vs. PBS-treated hypoxic group (One-way
ANOVA with Tukey-Kramer post-test).
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DOI: 10.1161/CIRCULATIONAHA.112.114173
Figure 7. MEX of either mouse or human origin suppress the hypoxic phosphorylation
(activation) of STAT3. (A) Total protein extracts from lungs of individual animals treated with
10 μg MEX. Hypoxia exposure for 2 days resulted in activation of STAT3 through
phosphorylation at Tyr-705 (pY-STAT3) and this was prevented by treatment with MEX of
mouse origin. Right panel: Quantitation of STAT3 phosphorylation. #, p<0.01 vs. PBS-treated
hypoxic group; ns, statistically non-significant (One-way ANOVA with Tukey-Kramer posttest). (B) Primary cultures of hPAECs exposed to hypoxia (1% O2, 6 hours) exhibit robust
activation of STAT3 that is efficiently suppressed by MEX secreted by MSCs from mouse
(mMEX)
mMEX) and human umbilical cord stroma (hMEX). Note that neither the microv
microvesicle-depleted
ovves
esic
icle
ic
lee-d
-dep
eple
ep
lete
le
t d
fraction of media conditioned by human umbilical cord MSCs (hUC-ExD-CM), nor hFEX or
mFEX,
mF
FEX
X, ha
have
ve aany
nyy eeffect
ffect on STAT3 phosphorylati
ff
phosphorylation.
tiion
on. Data are represen
representative
en
nta
tati
t ve of at least 3
ndeepe
p ndentt experiments.
expperi
ex
perime
ment
ntts.
independent
Figure
Figu
gure
re 88.. ME
M
MEX
X trea
treatment
eatm
tmentt suppresses
suupp
ppre
ress
s es the
he hypoxic
hyp
ypox
oxic
ic indu
induction
duct
ctio
ionn of the
he m
miR-17
iR-177 mi
iR
micr
microRNA
croR
oRNA
NA
upe
perf
rfam
amil
ilyy an
andd increases
incr
in
crea
ease
sess levels
leve
le
vels
ls of
of anti-proliferative
anti
an
ti-pro
proli
liffer
erat
ativ
ivee mi
miR
R-20
2044 in tthe
he llung.
ungg M
un
icee we
ic
were
re ttreated
reat
re
ated
ed
superfamily
miR-204
Mice
with either PBS, MEX (10 μg) or FEX (10 μg) and then exposed to hypoxia for 7 days. Another
untreated group remained in normoxia as control. MicroRNA levels in total mouse lung were
assessed by qPCR and fold changes in the hypoxic groups are presented relative to the mean of
the normoxic group. (A) Select miRs representing the miR-17~92, miR-106b~25 and miR106a~363 clusters. (B) Select miRs reported to be involved in hypoxic signaling. (C)
Upregulation of basal levels of the pulmonary arteriole-specific miR-204 upon MEX treatment.
Dots represent expression levels in individual animals. NRX : Normoxia; HPX : Hypoxia. ¶,
p<0.001 vs. normoxia; ‡, p<0.01 vs. normoxia; †, p
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Exosomes Mediate the Cytoprotective Action of Mesenchymal Stromal Cells on
Hypoxia-Induced Pulmonary Hypertension
Changjin Lee, S. Alex Mitsialis, Muhammad Aslam, Sally H. Vitali, Eleni Vergadi, Georgios
Konstantinou, Konstantinos Sdrimas, Angeles Fernandez-Gonzalez and Stella Kourembanas
Circulation. published online October 31, 2012;
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2012 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circ.ahajournals.org/content/early/2012/11/02/CIRCULATIONAHA.112.114173
Data Supplement (unedited) at:
http://circ.ahajournals.org/content/suppl/2012/10/30/CIRCULATIONAHA.112.114173.DC1.html
http://circ.ahajournals.org/content/suppl/2012/11/02/CIRCULATIONAHA.112.114173.DC2.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
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SUPPLEMENTAL MATERIAL
Expanded Methods
Animal model and hypoxic exposure
Eight to 10- week old mice (FVB strain) were housed in large plexiglass chambers and
the FiO2 regulated by an OxyCycler controller (BioSpherix, Redfield, NY). Hypoxic
exposures were performed at 8.5±0.5 % O2 and ventilation was adjusted to insure that
CO2 levels did not exceed 5,000 ppm (average range 1,000-3,000 ppm). Ammonia was
removed by ventilation and activated charcoal filtration through an air purifier. Under
these conditions, the ammonia concentration is less than 2.5 ppm, the limit of detection
of Gastec passive dosimetry tubes (Sigma). Animals were anesthetized with
pentobarbital (50 mg/kg, I.P.) and injected through the left jugular vein with concentrated
conditioned media (5 g protein in 50 l) or exosome preparations (0.1 g protein in 50
l PBS). A higher dose of exosomes (10 g protein in 50 l PBS) was delivered through
the tail vein. Table 1 lists the number of cells used and the exosomal protein recovery
from each cell type. Approximately 2% of secreted proteins in the conditioned media of
both mMSCs and MLFs are associated with the exosomal fraction and roughly 3-fold
more MLFs were required to extract equal amounts of exosomes for the injections. An
equal volume of PBS or serum-free -MEM media was injected in control experimental
groups. Mice were allowed to recover for 3 hours before placement in hypoxic
chambers. In certain time-course and dose-dependent studies, a second injection of
MEX (0.1 g protein in 50 l PBS) was performed on the contralateral jugular vein after
4 days of hypoxic exposure.
1
Isolation of human MSCs from umbilical cord Wharton’s Jelly
Human umbilical cord Wharton’s jelly derived MSCs (hUC-MSCs) were isolated
according to published methods1, 2 with minor modifications. Cord was rinsed twice with
cold sterile PBS, cut longitudinally, and arteries and vein were removed. The soft gel
tissues were scraped out, finely chopped (2-3 mm2) and directly placed on 100 mm
dishes (15 pieces per dish) with DMEM/F12 (1:1) (Invitrogen) supplemented with 10%
fetal bovine serum (Hyclone), 2 mM L-glutamine, and penicillin/streptomycin, and
incubated for 5 days at 37°C in a humidified atmosphere of 5% CO2. After removal of
tissue and medium, the plates were washed 3 times with PBS, the attached cells were
cultured and fresh media replaced 3 times per week. At 70-80% confluence, cells were
collected and stained with PE conjugated antibodies for CD34 (Miltenybiotec, Auburn,
CA) and CD45 (Miltenybiotec, Auburn, CA). Immunodepletion was performed using the
anti-PE-microbeads (Miltenybiotec, Auburn, CA) and MSCS column (Miltenybiotec,
Auburn, CA) according to manufacturer’s instructions. The CD34 and CD45 negative
populations were further propagated and selected for the expression of MSC markers
(CD105, CD90, CD44, and CD73) and the absence of CD11b, CD19, and HLA-DR by
using a set of fluorescently-labeled antibodies designed for the characterization of
human MSCs (BD Biosciences, San Diego, CA) and a MoFlo flow cytometry (Beckman
Coulter).
Cell culture and collection of conditioned media
Bone marrow-derived MSCs, isolated from the femurs and tibiae of 5-7 week old male
FVB mice, were selected and their differentiation potential assessed as previously
described3. Briefly, after 3-4 passages, plastic adherent cells were immunoselected
using mouse specific antibodies (BD Biosciences Pharmingen, San Diego, CA) and a
MoFlo fluorescence-activated cell sorter (FACS) (Dakocytomation, Fort Collins, CO), as
2
we reported previously3, 4 in compliance with published MSC criteria5. Cells were
negatively selected for CD11b, CD14, CD19, CD31, CD34, CD45, and CD79α antigens,
and positively selected for CD73, CD90, CD105, c-kit and Sca-1 antigens. Primary MLF
cultures were derived according to standard methods6, 7.
To exclude contamination from serum-derived microvesicles, serum used for
propagation of cell cultures and the collection of CM was clarified by ultracentrifugation
at 100,000 x g for 18 hours. MSCs were cultured in -MEM media supplemented with
10% (v/v) FBS (Hyclone), 10% (v/v) Horse Serum (Hyclone), 2 mM L-glutamine
(GIBCO), and antibiotics. MLFs were cultured in DMEM (Invitrogen) supplemented with
10% (v/v) FBS and 2 mM L-glutamine. Cultures at 70% confluence were washed twice
with PBS and incubated with serum-free media supplemented with 2 mM L-glutamine for
24 hours under standard culture conditions. Conditioned media were collected and cells
and debris were removed by differential centrifugations at 400 x g for 5 mins, at 2,000 xg
for 10 mins, and at 13,000 xg for 30 mins. The clarified CM were subsequently filtered
through a 0.2 m filter unit and concentrated using an Ultracel-10K (Millipore) centrifugal
filter device, to a protein concentration range of 0.1-0.5 mg/ml. Protein levels in the CM
were determined by Bradford assay (Bio-Rad, Hercules, CA).
Bronchoalveolar lavage
Animals were anesthetized with Avertin (250 mg/Kg i.p.) and their trachea cannulated
with a blunt-ended 20 gauge Luer Stub Adapter (Becton Dickinson). BALF was
collected via sequential administration of PBS supplemented with 5 mM EDTA (0.8 ml,
0.8 ml, 0.8 ml, and 0.9 ml) and approximately 3.0 ml (+/- 0.1 ml) of BALF was recovered
3
per animal. Cells in BALF were collected by centrifugation at 400 xg for 10 min and
leukocytes stained with Kimura solution8 for counting.
Right ventricular systolic pressure measurements
Mice were anesthetized with 60 mg/kg of pentobarbital and remained spontaneously
breathing. A small incision was made in the abdominal wall, and the translucent
diaphragm exposed. A 23-gauge butterfly needle with tubing attached to a pressure
transducer was inserted through the diaphragm into the right ventricle and pressure
measurements were recorded with PowerLab (ADInstruments, Colorado Springs, CO)
monitoring hardware and software. Animals with heart rates less than 300 beats per
minute were considered over-anesthetized and their RVSP measurements were
excluded. Mean RVSP over the first ten stable heartbeats was recorded.
Right ventricular weight measurements
Hearts and pulmonary vasculature were perfused in situ with cold 1X PBS injection into
the right ventricle; hearts were excised and used for Fulton’s Index measurements (ratio
of RV weight over left ventricle plus septal weight, RV/[LV+S]). Both ventricles were
weighed first, then the right ventricular free wall was dissected and the remaining LV and
ventricular septum was weighed.
Pulmonary histology
Lungs were inflated by tracheotomy and perfused with 4 % paraformaldehyde, excised,
and fixed in 4 % PFA overnight at 4oC followed by paraffin embedding. Sections (two per
animal) from 4 individuals in each group ( group n > 7) were analyzed for pulmonary
histology. For pulmonary vascular morphometry, paraffin-embedded lung sections were
stained with hematoxylin and eosin. For immunohistochemical analysis, 5 m lung
4
tissue sections were deparaffinized in xylene and rehydrated. Tissue slides were treated
with 0.3% H2O2 in methanol to inactivate endogenous peroxidases and blocked with
horse serum for 1 hour. After incubating with monoclonal anti-mouse -SMA antibody
(Sigma) at a dilution of 1:125 overnight at 4oC, secondary antibodies and peroxidase
staining was applied according to manufacturer's instructions (Vector Laboratories,
Burlingame, CA). Vessel wall thickness was assessed by measuring α-SMA staining in
vessels (20-40 m in diameter) within each field (40-50 fields per section) captured at
400X magnification with a microscope digital camera system (Nikon, Tokyo, Japan), and
using Metamorph image analysis program (Molecular Devices, Sunnyvale, CA). The
medial wall thickness index was calculated by the following formula: Wall thickness (%)
= 100 x (area[ext] – area[int]) / area[ext] where area[ext] and area[int] denote the areas
bounded by the SMA layer.
In vitro hypoxia
Human PAECs were purchased from GIBCO and cultured in M200 medium (Invitrogen)
supplemented with LSGS (Invitrogen). At 80% confluence, cells were exposed to 1% O2
for 6 hours in an inVivo2 workstation (Ruskin Technology, Bridgend, UK) in the presence
or absence of exosomal fraction (1 g/ml), or the exosome-depleted fraction of hUCMSC conditioned media (1 g/ml). Cells were lysed and proteins in whole cell lysates
were separated on 8% SDS-polyacrylamide gel electrophoresis followed by western blot
analysis using rabbit monoclonal antibody for phospho-STAT3 (Y705) and mouse
monoclonal STAT3 antibody (Cell Signaling).
5
Electron microscopic analysis
EM analysis was performed at the Harvard Medical School electron microscope facility.
Exosome preparations were adsorbed to a carbon coated grid that had been made
hydrophilic by a 30 second exposure to a glow discharge. Excess liquid was removed
and the samples were stained with 0.75% uranyl formate for 30 seconds. After removing
the excess uranyl formate, the grids were examined in a JEOL 1200EX Transmission
electron microscope and images were recorded with an AMT 2k CCD camera.
Protein extraction and immunoblotting
BALF (3 ml) was centrifuged at 420 x g for 10 min and cell-free BALF supernatants were
used for protein analysis. Equal volumes of BALF specimens from individual animals in
the same group were pooled (1 ml) and proteins precipitated overnight by 20%
trichloroacetic acid (Sigma). A fraction equivalent to 30% of each protein pellet was
dissolved in 1x sodium lauryl sulfate (SDS)-loading buffer was separated on a
denaturing 15% polyacrylamide gel. After transfer to 0.2 m PVDF membranes
(Millipore), blots were blocked with 5% skim milk and incubated with 1:1,000 diluted
rabbit polyclonal MCP-1, galectin-3, or HIMF/FIZZ1 antibody (Abcam) for overnight at
4C. To detect mouse Immunoglobulin A, 1:5,000 diluted goat anti-mouse IgA antibody
(Abcam) was used. Peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz
Biotech) was used in 1:20,000 dilution to visualize immunoreactive bands either by the
enhanced chemiluminescence reagent (Pierce) or Lumi-LightPLUS (Roche).
For analysis of proteins from whole lung, frozen lung tissues were homogenized for 5
seconds with Polytron in cold PBS containing 2 mM phenylmethanesulfonyl fluoride
6
(Sigma) and centrifuged at 3,000 xg for 3 mins. Tissue pellets were washed twice with
cold PBS containing 2 mM PMSF followed by centrifugation at 3,000 xg for 3 mins and
lysed in RIPA buffer containing protease inhibitor (Roche) and phosphatase inhibitor
cocktails (Thermo). Forty g of lung tissue extracts were separated on 10-20% gradient
gel (Invitrogen). Antibodies for MCP-1, HIMF, IL-6, STAT3, and phospho-STAT3 (Y705)
were used for immunoblotting. For loading control, mouse monoclonal -actin antibody
(Sigma) was used.
Proteins in exosome preparations were separated on 12% polyacrylamide gel and then
transferred onto 0.45 m PVDF membrane (Millipore). Goat polyclonal anti-CD63
antibody (Santa Cruz Biotech), mouse monoclonal Alix and TSG101 antibodies (Santa
Cruz Biotech), rabbit polyclonal CD81, CD9, hsp90, and flotillin-1 antibodies (Santa Cruz
Biotech), and rabbit polyclonal Dicer (Abcam) antibody were used for immunoblotting.
Isolation and Quantification of microRNAs
Total lung RNA was extracted by the method of Chomczynski & Sacchi9 and 750 ng was
used as a template for reverse transcriptase with specific primers for each target
microRNA (TaqMan Reverse Transcription Kit, Applied Biosystems, Foster City, CA).
Each reverse transcription reaction included also the primer for the small nuclear RNA
sno202, which was used as an internal control. 37.5 ng cDNA was used for each 20 l
qPCR reaction with TaqMan universal master mix II with no UNG (Applied Biosystems)
in the presence of probes specific for the indicated microRNAs and the internal control
(TaqMan microRNA assay, Applied Biosystems). Amplification was performed at 50oC
for 2 min, 95oC for 10 min, followed by 40 cycles of 95oC for 15 sec, 60oC for 1 min, on a
StepOne Plus platform (Applied Biosystems).
7
RNAs from isolated exosomes were extracted by Trizol reagent (Invitrogen). Briefly, 30
µg exosomal protein was mixed with 0.5 ml Trizol reagent per manufacturer’s
recommendation. 20 µg RNase-free glycogen (Ambion) was applied as a carrier prior to
RNA precipitation with isopropyl alcohol and samples were placed at -80 °C for
overnight. 150 ng exosomal RNAs were used as a template in reverse transcription
reactions with specific primers for target microRNAs (TaqMan Reverse Transcription Kit,
Applied Biosystems). To quantify pre-let7b, 300 ng of exosomal RNAs were reverse
transcribed by High Capacity RNA-to-cDNA kit (Applied Biosystems) per manufacturer’s
recommendations. 7.5 ng of cDNA for each microRNA assay and 11.5 ng of cDNA for
pre-let7b (TaqMan gene expression assay, Applied Biosystems) were used for qPCR
reaction in the presence of specific probes. Amplification was performed as described
above. Let7a was used as an internal control.
Smooth Muscle Cell Proliferation Assay
Primary rat PASMCs were inoculated at a concentration of 2 x 103/well on a 96 well plate
in DMEM containing 5% FBS and incubated for 24 hours under standard culture
conditions. After serum starvation for 2 days in 0.1% FBS/DMEM, cells were pretreated
either with vehicle or varying doses of mMEX (16, 31.5, 62.5, 125 ng/ml) for 30 min then
FBS was added at 5% (v/v) to each well. After incubation for 48 hours, cell proliferation
reagent WST-1 (Roche), which is cleaved by mitochondrial dehydrogenases in
metabolically active cells to form formazan dye, was directly applied to the cells followed
by further incubation for 3 hours. Intensity of solubilized dark red formazan was
determined at 440 nm using a microplate reader.
8
Supplemental References
1.
Mitchell KE, Weiss ML, Mitchell BM, Martin P, Davis D, Morales L, Helwig B,
Beerenstrauch M, Abou-Easa K, Hildreth T, Troyer D, Medicetty S. Matrix cells
from Wharton's jelly form neurons and glia. Stem Cells. 2003;21:50-60.
2.
Penolazzi L, Mazzitelli S, Vecchiatini R, Torreggiani E, Lambertini E, Johnson S,
Badylak SF, Piva R, Nastruzzi C. Human mesenchymal stem cells seeded on
extracellular matrix scaffold: Viability and osteogenic potential J Cell Physiol.
2011;[published online ahead of print August 9, 2011] doi: 10.1002/jcp.22983.
3.
Aslam M, Baveja R, Liang OD, Fernandez-Gonzalez A, Lee C, Mitsialis SA,
Kourembanas S. Bone marrow stromal cells attenuate lung injury in a murine
model of neonatal chronic lung disease. Am J Respir Crit Care Med.
2009;180:1122-1130.
4.
Liang OD, Mitsialis SA, Chang MS, Vergadi E, Lee C, Aslam M, FernandezGonzalez A, Liu X, Baveja R, Kourembanas S. Mesenchymal stromal cells
expressing heme oxygenase-1 reverse pulmonary hypertension. Stem Cells.
2011;29:99-107.
5.
Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D,
Deans R, Keating A, Prockop D, Horwitz E. Minimal criteria for defining
multipotent mesenchymal stromal cells. The International Society for Cellular
Therapy position statement. Cytotherapy. 2006;8:315-317.
6.
McGowan SE. Extracellular matrix and the regulation of lung development and
repair. FASEB. 1992;6:2895-2904.
7.
McGowan SE. Influences of endogenous and exogenous TGF-beta on elastin in
rat lung fibroblasts and aortic smooth muscle cells. Am J Physiol.
1992;263:L257-263.
9
8.
Kimura I, Moritani Y, Tanizaki Y. Basophils in bronchial asthma with reference to
reagin-type allergy. Clin Allergy. 1973;3:195-202.
9.
Chomczynski P, Sacchi N. Single step method of RNA isolation by guanidinium
thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987;162:156-159.
10
11
Supplemental Figure Legends
Supplemental Figure 1
Flow cytometric analysis of surface-markers for human MSCs. Human MSCs from
umbilical stroma (hUC-MSCs) were cultured in DMEM/F-12(1:1) supplemented with 10%
FBS. Human UC-MSCs at passage 5 express CD90, CD105, CD73, CD44, but lack
expression of CD19, CD34, CD45, CD11b, and HLA-DR in flow cytometric analysis.
Isotype-matched IgG controls are shown with non-shaded dotted curves, and hUCMSCs curves are shown in red shaded area.
Supplemental Figure 2
Comparison of exosomes from two methods of isolation. Exosomes in the medium
conditioned by mMSCs were isolated by ultracentrifugation (UCF) or S200 sizeexclusion chromatography (SEC). Two g of proteins from each preparation were loaded
onto 12% SDS-PAGE and total proteins stained by SimplyBlue (Invitrogen) (left panel).
Exosomal markers, HSP90, flotillin-1, and CD63 were detected by immunoblotting and
are comparable in both preparations (right panel).
Supplemental Figure 3
Dose-dependent inhibition of SMC proliferation by MEX. Cultured rat PASMCs were
serum-deprived for 48 hours followed by treatment with mMEX (16 to 125 ng/ml) in the
presence of 5% FBS and their proliferation rate was quantified relative to the treatment
with FBS alone. Data are expressed as mean values ± SD. *, p < 0.001 vs. FBS-alone
(One-way ANOVA with Tukey-Kramer post-test).
12
Supplemental Figure 4
MicroRNA content in mouse MEX compared with FEX. RNAs extracted from equivalent
amount of MEX and FEX were subjected to RT-qPCR analysis. Levels of the indicated
microRNAs relative to let7a in MEX and FEX are presented on the left panel and
comparisons between the level of pre-let7b and let7b relative to let7a in MEX and FEX
are shown on the right. Data are presented as mean values ±SD. *, p < 0.001 MEX vs.
FEX (Student’s t-test).
Supplemental Figure 5
Schema of a hypothesis synthesizing the results of this study with our previous work and
published literature. Hypoxia shifts the Th1/Th2 balance of immunomodulators in the
lung, resulting in alternative activated alveolar macrophages (AA-AM) and, in the early
phase, induces the expression of IL-6, MCP-1, and HIMF in the lung epithelium. HIMF
mitogenic action on the vasculature requires Th2 cytokines, such as IL-4. Consequences
of the shift towards proliferation include the hypoxic activation of STAT3 signaling and
the upregulation of the miR-17 family of microRNAs. Treatment with MEX interferes with
an early hypoxic signal in the lung, suppressing both inflammation and HIMF
transcriptional upregulation. It addition, MEX treatment may directly upregulate miR-204
levels, thus breaking the STAT3-miR-204-STAT3 feed-forward loop, and shifting the
balance to an anti-proliferative state.
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15
16
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18
Correction
In the article by Lee et al, “Exosomes Mediate the Cytoprotective Action of
Mesenchymal Stromal Cells on Hypoxia-Induced Pulmonary Hypertension,” which was
published ahead of print on October 31, 2012, an error occurred.
In the initial publication of the article, Figure 6 was incorrect. The error has been
corrected in the current online version of the article.
The Editorial Office regrets the error.