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Fu in deficiency in myeloid cells leads o a enua ed e ascula iza ion in a
mouse-model o oxygen-induced e inopa hy
Ma ia Vähä upa
a
, Zuze Ma inez Co do a
a,b
, Ha lan Ba ke
a
, Saa a Ai omäki
a,b
,
Hannu Uusi alo
a,c,∗
, Te o A.H. Jä inen
a,d
, Ma ko Pesu
a,b,e
, Hannele Uusi alo-Jä inen
a,c
a
Facul y o Medicine & Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland
b
Immuno egula ion, Ins i u e o Biosciences and Medical Technology (BioMediTech), Uni e si y o Tampe e, Tampe e, Finland
c
Eye Cen e, Tampe e Uni e si y Hospi al, Tampe e, Finland
d
Depa men s o Musculoskele al Diso de s, Tampe e Uni e si y Hospi al, Tampe e, Finland
e
Depa men s o De ma ology, Tampe e Uni e si y Hospi al, Tampe e, Finland
ARTICLE INFO
Keywo ds:
Angiogenesis
Fu in
Mac ophage
Oxygen-induced e inopa hy model
Hypoxia
ABSTRACT
Ischemic e inopa hy is a ision- h ea ening disease associa ed wi h ch onic e inal inflamma ion and hypoxia
leading o abno mal angiogenesis. Fu in, a membe o he p op o ein con e ase amily o p o eins, has been
implica ed in he egula ion o angiogenesis due o i s essen ial ole in he ac i a ion o se e al angiogenic
g ow h ac o s, including ascula endo helial g ow h ac o -C (VEGF-C), VEGF-D and ans o ming g ow h
ac o - β(TGF- β). In he p esen s udy, we e alua ed exp ession o u in in he e ina and i s ole in e inal
angiogenesis. As bo h inflamma ion and hypoxia con ibu e o angiogenesis, he ole o u in was e alua ed
using myeloid-cell specific u in knockou (KO) mice (designa ed LysMC e- u
(fl/fl)
) bo h in de elopmen al e -
inal angiogenesis as well as in hypoxia-d i en angiogenesis using he oxygen-induced e inopa hy (OIR) model.
In he e ina, u in exp ession was de ec ed in endo helial cells, mac ophages and, o some ex en , in neu ons.
The a e o angiogenesis was no diffe en in LysMC e- u
(fl/fl)
mice when compa ed o hei wild- ype li e ma es
du ing de elopmen . In he OIR model, he e ascula iza ion o e ina was significan ly delayed in LysMC e-
u
(fl/fl)
mice compa ed o hei wild- ype li e ma es, while he e was no compensa o y inc ease in he p e e inal
neo ascula iza ion in LysMC e- u
(fl/fl)
mice. These esul s demons a e ha u in exp ession in myeloid cells
plays a significan ole in hypoxia-induced angiogenesis in e ina.
1. In oduc ion
Ischemic e inopa hies, such as e inopa hy o p ema u i y (ROP),
p oli e a i e diabe ic e inopa hy (PDR), and e inal ein occlusion
(RVO) a e majo causes o isual impai men and blindness in in-
dus ialized coun ies (Fong e al., 2004; Jonas e al., 2017; Reynolds,
2014). Re inal ischemia induces he s abiliza ion o a ansc ip ion
ac o hypoxia inducible ac o -1α(HIF-1α), which u ns on he ex-
p ession o a la ge numbe o angiogenic genes such as ascula en-
do helial g ow h ac o (VEGF) (Campochia o, 2015). This leads o
e inal neo ascula iza ion associa ed wi h i eous hemo hages and
fib osis o neo ascula memb anes causing ac ional e inal de ach-
men and, ul ima ely, blindness (Campochia o, 2015).
Re inal ischemia is also associa ed wi h inflamma o y mani es a-
ions. Hypoxia a ac s mac ophages in o hypoxic a eas (Mi amu a
e al., 2005; Tsu sumi e al., 2003) whe e he hypoxia-ac i a ed mac-
ophages and mic oglia, he immune cells o he e ina, elease no only
p oinflamma o y bu also angiogenic cy okines. In diabe ic e inopa hy
(DR), bo h in ading mac ophages and esiden mic oglia ha e been
implica ed in he pa hogenesis o he e inopa hy e inopa hy
(Abcouwe , 2012). O no e, mac ophages ha e been shown o be he
main sou ce o VEGF-C in he e ina. In he e ina, VEGF-C signaling
pa hway egula es he b anching o blood essels (Tammela e al.,
2011). VEGF-C is ini ially syn hesized as an inac i e p ecu so and e-
qui es ac i a ion by a p op o ein con e ase sub ilisin/kexin (PCSK)
amily membe u in o become biologically ac i e (Sieg ied e al.,
2003b).
P op o ein con e ase sub ilisin/kexin (PCSK) amily enzymes a e a
amily o nine p o eases (PCSK1-2, u in, PCSK4-7, MBTPS1, PCSK9)
ha clea e and con e hei imma u e a ge p o eins in o biologically
ac i e o ms by ca alyzing endop o eoly ic clea age a a a ge si e
ypically made up o he basic amino acids a ginine and lysine
(Tu peinen e al., 2013). Acco dingly, PCSK enzymes play a key eg-
ula o y ole in a mul i ude o biological e en s go e ned by he g ow h
h ps://doi.o g/10.1016/j.exe .2017.10.013
Recei ed 4 Janua y 2017; Recei ed in e ised o m 1 Sep embe 2017; Accep ed 11 Oc obe 2017
∗
Co esponding au ho Facul y o Medicine & Li e Sciences, 33014, Uni e si y o Tampe e, Finland.
E-mail add ess: llhauus@u a.fi(H. Uusi alo).
Expe imen al Eye Resea ch 166 (2018) 160–167
A ailable online 13 Oc obe 2017
0014-4835/ © 2017 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/BY-NC-ND/4.0/).
T
ac o s (Tu peinen e al., 2013). Among he PCSK amily membe s,
u in is a ubiqui ously exp essed p o o ypical se ine endop o ease e-
cen ly implica ed o play a key ole in a ious biological p ocesses such
au oimmuni y and inflamma ion, and a ious human diseases (Oksanen
e al., 2014; Pesu e al., 2008; Tu peinen e al., 2013; Vähä upa e al.,
2016a). In fib o ascula issue om PDR pa ien s, u in exp ession was
shown o co-localize wi h one o i s clea age a ge s, a (p o)- enin e-
cep o p o ein, whose exp ession le el is ele a ed in PDR and asso-
cia ed wi h angiogenic ac i i y, sugges ing ha u in clea age has a ole
in DR (Kanda e al., 2012).
In addi ion o VEGF-C (Kha ib e al., 2010; Sieg ied e al., 2003a),
u in is also equi ed o he ac i a ion o a la ge numbe o o he
p oangiogenic g ow h ac o s, such as VEGF-D (McColl e al., 2007),
pla ele de i ed g ow h ac o s A and B (PDGF-A and –B) (Sieg ied
e al., 2003b, 2005), bone mo phogene ic p o ein-4 (BMP-4) (Cui e al.,
1998) and ans o ming g ow h ac o -β(TGF-β)(Dubois e al., 2001).
On he o he hand, he exp ession o u in i sel can be induced by
hypoxia, as i is one o he a ge genes o HIF-1α(Ma e al., 2017;
McMahon e al., 2005; Sil es i e al., 2008). Despi e being linked o
angiogenesis, he unc ions o u in in hese p ocesses a e s ill poo ly
known, bu he pheno ype o he o al u in knockou (KO) mice,
le hali y du ing emb yonal de elopmen due o ca dio ascula de ec s,
sugges ed blood essel specific unc ions (Roeb oek e al., 1998;
Scamuffa e al., 2006). This no ion was confi med by endo helial spe-
cific u in KO ha also led o emb yonal dea h due o ascula de ec s,
and by he endo helial cells lacking u in ha we e unable o g ow
ex i o (Kim e al., 2012).
As endo helial cells lacking u in canno g ow a all (Kim e al.,
2012), we decided o elucida e he ole o u in on e inal angiogenesis
by add essing he exp ession o u in in he e ina and by assessing he
effec o myeloid cell-de i ed u in on e inal angiogenesis using u in
condi ional KO mice (myeloid cell-specific u in KO; LysMC e- u
(fl/fl)
).
The effec o myeloid cells-de i ed u in on angiogenesis was s udied in
de elopmen al e inal angiogenesis, as well as in hypoxia-induced an-
giogenesis in a mouse-model o oxygen-induced e inopa hy (OIR),
which is he mos widely used model o ischemic e inopa hies
(Conno e al., 2009; Smi h e al., 1994). In he OIR model, bo h e-
c ui ed, blood-de i ed mac ophages as well as he esiden mac o-
phages o he e ina, mic oglia, ha e been shown o pa icipa e in he
egula ion o angiogenesis (Checchin e al., 2006; Gao e al., 2016;
Ka aoka e al., 2011; Zhou e al., 2015).
2. Resul s
2.1. Fu in is exp essed in mac ophages and e inal mic oglia
Using da a om published genome-wide exp ession s udies, he
e inal-cell specific E-GEOD-33089 expe imen and he much wide
sea ching FANTOM p ojec , we ex ac ed exp ession da a o u in in
cells ela ed o e ina. In he la ge , bu a less specific, FANTOM
p ojec cDNA analysis o u in exp ession showed he highes alues in
monocy e de i ed mac ophages (Fig. 1A). In he e inal cell-specific
expe imen , based on 155 samples analyzed by mic oa ay, he highes
u in exp ession was ound in mic oglia (Fig. 1B).
2.2. Fu in is exp essed in he e ina du ing de elopmen and in he OIR
model
To u he s udy he ole o u in on e inal angiogenesis in i o,we
fi s de e mined a empo al and spa ial exp ession p ofile o u in in WT
mice du ing de elopmen and in he OIR model. Immunohis ochemis y
showed a ew cells wi h weak exp ession o u in in e ina a P4
(pos na al day 4), and he ea e he exp ession o u in was sub-
s an ially s onge om P7 onwa ds a all s udied ime poin s du ing
de elopmen (Fig. 1C). In he OIR model, s ong exp ession o u in was
de ec ed in he ascula laye s o he e ina a P14 and P17 (Fig. 1D).
Using double-immunohis ochemis y o u in and o endo helial cells
ma ke CD31 as well as o u in and mac ophage ma ke F4/80, u in
exp ession was localized o endo helial cells (Fig. 1E–F) in he e ina
and in he p e e inal ascula u s as well as o mac ophages su -
ounding blood essels (Fig. 1G–H) in OIR e inas. In addi ion o hese
cells, u in immuno eac i i y was also de ec ed in neu ons in he e ina
(Fig. 1).
2.3. Lack o u in in myeloid cells does no influence he a e o
de elopmen al angiogenesis
To in es iga e he ole o u in in angiogenesis, we s udied mice
deficien o u in gene exp ession in myeloid cells (LysMC e- u
(fl/fl)
)
and hei espec i e wild- ype li e ma es (LysM WT) du ing de elop-
men al e inal angiogenesis and du ing ischemia induced e inal an-
giogenesis using oxygen-induced e inopa hy model (OIR). LysMC e-
u
(fl/fl)
mice a e e ile wi hou no ob ious mo phological abno mal-
i ies (Co do a e al., 2016), and hei eyes appea no mal upon his o-
logical examina ion. As o al deple ion o esiden e inal mac ophages
has been shown o slow down he a e o de elopmen al e inal an-
giogenesis (Checchin e al., 2006), we fi s explo ed whe he myeloid
cell de i ed u in influences he a e o supe ficial ascula plexus
o ma ion (de elopmen al angiogenesis) in e inas om P6 LysM WT
and LysMC e- u
(fl/fl)
. In P6 neona al mice, he diame e s o e inal
supe ficial ascula plexuses we e simila and no diffe ences in ei he
he numbe o b anching poin s o filopodia coun we e de ec ed be-
ween LysMC e- u
(fl/fl)
and LysM WT mice, indica ing ha u in ex-
p ession by myeloid cells does no ha e a majo influence on in i o
angiogenesis du ing neona al de elopmen (Fig. 2A–E). This finding is
in line wi h he abo e epo ed appea ance o u in a he la e s ages o
e inal ascula de elopmen (Fig. 1C).
2.4. Mice deficien o u in exp ession in myeloid cells show educed
hypoxic e ascula iza ion a e o he e ina
The ole o myeloid cell de i ed u in in hypoxia-d i en neo ascu-
la iza ion in he e ina was explo ed nex . Fi s , we de e mined whe he
eg ession o essels unde hype oxic condi ions in he OIR model is
compa able be ween LysMC e- u
(fl/fl)
and LysM WT mice. A e ex-
posu e o 75% oxygen be ween P7 and P12, e inas we e e alua ed a
P12. Typical o his model (Smi h e al., 1994; Uusi alo-Jä inen e al.,
2007; Vähä upa e al., 2016b), la ge a eas o he cen al ascula ne -
wo k we e eg essed wi h only a ew majo essels emaining cen ally
in bo h LysM WT and LysMC e- u
(fl/fl)
mice (Fig. 2F). Quan i a i e
analysis o aso-obli e a ed a eas in e ina confi med ha he e inal
ascula u e in LysMC e- u
(fl/fl)
and LysM WT mice was simila ly a -
ec ed by hype oxic exposu e (Fig. 2G). Taken oge he , hese da a
sugges ha dele ion o u in om myeloid cells has no a significan
effec on ascula eg ession in OIR model.
On e u n o no moxia, he a ascula cen al e ina becomes hy-
poxic and s imula es apid eg ow h o essels (Smi h e al., 1994;
Uusi alo-Jä inen e al., 2007). The a e o e inal e ascula iza ion in
LysM WT and LysMC e- u
(fl/fl)
e inas was de e mined by quan i ying
he a ascula e inal a ea 5 days a e he mice we e e u ned o no -
moxia. The e was a s a is ically significan diffe ence in he a ascula
e inal a ea be ween he LysM WT and LysMC e- u
(fl/fl)
mice a P17
showing ha he a e o e inal e ascula iza ion is subs an ially (30%)
educed in mice whe e u in is deple ed om myeloid cells (Fig. 2J). In
addi ion o e ascula iza ion o he e ina, he s ong hypoxic s imulus
om he cen e o he e ina also d i es abno mal misdi ec ed sp ou ing
o blood essels owa ds he i eous a he in e ace be ween he
cen ally obli e a ed and pe iphe ally pe used e ina (Smi h e al.,
1994; Uusi alo-Jä inen e al., 2007). This pa hological, p e e inal
neo ascula iza ion eaches i s maximum 5 days a e e u n o no -
moxia (a P17). The e was no s a is ically significan diffe ence in he
amoun o p e e inal neo ascula iza ion be ween LysM WT and
M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167
161
(cap ion on nex page)
M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167
162
LysMC e- u
(fl/fl)
e inas (Fig. 2K). The esul s indica e ha myeloid cell
exp essed u in has a ole in hypoxia-induced e ascula iza ion in e-
ina, bu does no influence he pa hological p e e inal neo ascula -
iza ion.
3. Discussion
The p esen s udy demons a es ha mac ophages associa ed wi h
e inal blood essels exp ess u in and he lack o u in in myeloid cells
delays hypoxia-d i en angiogenesis in e ina. I has p e iously been
shown ha he endo helial specificdeficiency o u in leads o emb y-
onal dea h due o ascula de ec s (Roeb oek e al., 1998), and he
cul u ed endo helial cells lacking u in canno g ow (Kim e al., 2012).
Ou esul s confi m an impo an ole o u in in angiogenesis as we
show o he fi s ime ha u in deficiency in myeloid cells leads o
impai ed angiogenesis in hypoxia-induced e inal angiogenesis.
P e ious s udies ha e shown ha esiden mac ophages, mic oglia,
a e needed o p ope angiogenesis in e ina du ing bo h de elopmen al
and hypoxia-induced angiogenesis (Checchin e al., 2006). In his s udy,
we demons a e ha myeloid-cell specific dele ion o p op o ein con-
e ase u in leads o he a enua ion o angiogenesis and educed
ascula iza ion a e in OIR model. Among u in's a ge p o eins a e
se e al p oangiogenic g ow h ac o s, including VEGF-C which is ex-
p essed in mac ophages and has a ole in con olling he b anching o
blood essels in he e ina (Sieg ied e al., 2003a; Tammela e al.,
2011). VEGF-A, in u n, does no equi e ac i a ing clea age by u in.
In e es ingly, a myeloid-cell specific abla ion o VEGF-A exp ession
does no change he VEGF le els in he OIR and does no ha e any
influence on angiogenesis in he OIR model (Liyanage e al., 2016).
Al hough u he s udies conce ning he ole o u in in e inal angio-
genesis a e equi ed, ou esul s sugges ha he exp ession o u in in
mac ophages may be ela ed o ac i a ion o p oangiogenic g ow h
ac o s by u in in he e ina. This is in line wi h p e ious s udies
showing simul aneous induc ion o VEGF-C and u in exp ession in
diffe en disease models (Kha ib e al., 2010; Lopez de Cicco e al.,
2004; Sieg ied e al., 2003a).
A majo unc ion o u in is o con ol he bioa ailabili y o an i-
inflamma o y TGF-β1(Pesu e al., 2008). As a ma e o ac , bo h u in
and TGF-βa e in ol ed in he egula ion o each o he 's ac i i y; u in is
needed o he ac i a ion o diffe en TGF-βiso o ms om inac i e
p ecu so molecules o ac i e, ma u e o ms (Pesu e al., 2008; Ven u a
e al., 2017). The ac i e TGF-β2, in u n, induces u in exp ession
(Ven u a e al., 2017). Thus, oge he hey gene a e a sel -sus aining
loop o high TGF-βac i i y (Ven u a e al., 2017). TGF-βsignaling is
associa ed wi h neo ascula iza ion in many diseases, including neo-
ascula ocula diseases (Amin e al., 1994; Bai e al., 2014; Wang e al.,
2017). The exp ession o TGF-βis ele a ed in he OIR model, sugges ing
ha TGF-βsignaling con ibu es o e inal e ascula iza ion
(Yingchuan e al., 2010). A p e ious s udy has shown ha he TGF-β1
bioac i i y is educed in mice deficien in u in exp ession in myeloid
cells (Co do a e al., 2016). Thus, ano he plausible explana ion o he
educ ion o e ascula iza ion in he e ina o LysMC e- u
(fl/fl)
mice
could be he educed bioa ailabili y o TGF-βdue o lack o ac i a ing
u in.
Al hough e inal e ascula iza ion a e and he o ma ion o pa-
hological p e e inal u s a e in e - ela ed in he OIR model, we
iden ified a delayed e ascula iza ion a e in e ina wi hou a sub-
sequen inc ease o pa hological p e e inal u o ma ion in LysMC e-
u
(fl/fl)
mice in he OIR model. One explana ion is ha esiden mac-
ophages, mic oglia, pe sis in he e ina in esponse o fi e-day long
hype oxia despi e blood essels disappea ing comple ely om e ina
(Da ies e al., 2006). Once he e ascula iza ion is ini ia ed upon e u n
o no moxia, i.e. induc ion o hypoxia in e ina, esiden mic oglial cells
espond o hypoxia by sec e ing p oangiogenic g ow h ac o s and di-
ec he e ascula iza ion, bu selec i ely only in e ina, whe e hey a e
loca ed. This no ion is suppo ed by he ac s ha he mic oglia a e
mainly loca ed in he a ascula a eas a e he hype oxia-pe iod and he
unc ion o mac ophage-de i ed VEGF-C is o con e he ip cells o
s alk cells du ing e inal angiogenesis (Tammela e al., 2011). The lack
o influence on p e e inal pa hological e ascula iza ion in he
LysMC e- u
(fl/fl)
mice is a he s iking because we obse ed sig-
nifican ly la ge a ascula a eas in LysMC e- u
(fl/fl)
mice han in WT
mice. This, in u n, should heo e ically induce mo e p onounced
compensa o y p e e inal pa hological u o ma ion, bu we could no
demons a e ha . An explana ion why la ge a ascula e inal a ea
does no ansla e in o enhanced p e e inal neo ascula iza ion, may be
impai ed p oangiogenic g ow h ac o ac i a ion in LysMC e- u
(fl/fl)
mice.
Taken oge he ou s udy demons a es ha he lack o u in in
myeloid cells delays hypoxia-d i en angiogenesis in e ina and im-
plica es u in exp essed ou side o he endo helial cells o he egula ion
o hypoxia-induced angiogenesis.
4. Me hods
4.1. Mice
Monocy e/Mac ophage-specific u in condi ional knockou
LysMC e- u
(fl/fl)
was gene a ed as desc ibed p e iously (Co do a e al.,
2016). B iefly, mice bea ing floxed u alleles we e backc ossed six
imes wi h C57BL/6 mice. LysMC e mice on C57BL/6 backg ound we e
pu chased om Taconic. LysMC e mice we e b ed wi h u
(fl/fl)
animals
o gene a e myeloid-specific u in knockou mice LysMC e- u
(fl/fl)
.
Mice we e housed unde pa hogen- ee s anda d condi ions, b ed and
he geno ype was de e mined by PCR. Mice we e ed wi h s anda d
labo a o y pelle s and wa e ad libi um. All animal expe imen s we e
pe o med acco ding o he ARVO s a emen o he use o animals in
oph halmic and ision esea ch in acco dance wi h p o ocols app o ed
by he Na ional Animal E hics Commi ee o Finland.
4.2. Oxygen-induced e inopa hy (OIR) model
The expe imen s on OIR model we e ca ied ou as desc ibed in
de ail p e iously (Smi h e al., 1994; S ahl e al., 2010b; Uusi alo-
Jä inen e al., 2007; Vähä upa e al., 2016b). B iefly, neona al mice a
P7 and hei nu sing mo he we e exposed o 75% oxygen o 5 days. A
P12, he mice we e e u ned o no mal oom ai . Animals we e eu-
hanized a P12 o assess he deg ee o ascula eg ession and a P17 o
de e mine he a e o e inal e ascula iza ion and p e e inal neo-
ascula iza ion. As pos na al weigh gain has been shown o affec
ou come in he OIR model, he pups included in he s udy we e weigh -
ma ched (S ahl e al., 2010a).
Fig. 1. Fu in is exp essed by he endo helial cells and mac ophages in e ina. Using exp ession da a om he FANTOM 5 p ojec , cDNA exp ession da a was ex ac ed o he p ima y
u in CAGE o ele an cell ypes (A). Exp ession da a o u in in diffe en e inal cell ypes was ex ac ed om p e iously pe o med mic oa ay exp ession analysis (B). In panels A–B
he cell ypes a e so ed by inc easing a e age exp ession om le o igh . Fu in exp ession om e ina was de e mined du ing de elopmen al angiogenesis a P4, P7 and P14 and in OIR
model a P14 and P17 om he e ascula ized e inas by IHC and IF using u in specific an ibody. Rep esen a i e images o u in exp ession du ing de elopmen (C) and in he OIR model
(D). A ows a e poin ing u in specific s aining a P17 OIR. Scale ba s ep esen s 100 μm. Rep esen a i e con ocal images showing CD31 posi i e endo helial cells ( ed) and u in (g een)
(E–F) and F4/80 posi i e mac ophages ( ed) and u in (g een) (G–H) in he e ina o ozen sec ions a e immunofluo escence s aining. Panels E and G a e showing s ainings a 20×
magnifica ion and panels F and H a 63× magnifica ion. A ows a e poin ing he endo helial cells o mac ophages ha a e exp essing u in. A owheads a e poin ing neo ascula u s.
GCL, ganglion cell laye ; IPL, inne plexi o m laye ; INL, inne nuclea laye ; OPL, ou e plexi o m laye ; ONL, ou e nuclea laye ; IS/OS, pho o ecep o inne /ou e segmen s; RPE,
e inal pigmen epi helium. Scale ba s ep esen s 100 μm in E and G and 20 μm in F and H.
M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167
163
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M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167
164

4.3. FANTOM analysis o e inal cell ypes
The FANTOM p ojec has pe o med exp ession analysis on 1839
samples om 573 p ima y cells, 152 issues, and 250 cell lines in
human. Specifically, he da a comes om cap analysis gene exp ession
(CAGE) sequencing o cDNA (FANTOM Conso ium e al., 2014). Ex-
p ession da a was ex ac ed o he p ima y u in CAGE o ele an cell
ypes: endo helial cells, pe icy es, as ocy es, e inal pigmen epi he-
lium cells, neu ons, neu ophils, endo helial p ogeni o cells, co neal
epi helial cells and monocy e de i ed mac ophages.
4.4. Mic oa ay analysis o e inal cell ypes
A mic oa ay exp ession analysis o mouse genes in a ious e inal
cell ype g oups was pe o med p e iously and is s o ed as accession E-
GEOD-33089 on he A ayExp ess da abase (h ps://www.ebi.ac.uk/
a ayexp ess/expe imen s/E-GEOD-33089). F om he da a we ex-
ac ed u in gene exp ession alues o 155 samples ac oss 7 cell ype
g oups: amac ine, cone, od, bipola , ho izon al, ganglion, and mic o-
glia (Siege e al., 2012). Bo h FANTOM and mic oa ay analyses we e
pe o med u ilizing supe compu e esou ces p o ided by CSC–IT
Cen e o Science o he Finnish Minis y o Educa ion and Cul u e and
he aw da a was p ocessed as p e iously desc ibed in de ail elsewhe e
(Ba ke e al., 2017).
4.5. Immunohis ochemis y (IHC) and isolec in GS-IB
4
s aining
Fo immunohis ochemis y, he eyes we e fixed wi h 4% PFA and
embedded in pa affin ( o IHC), o eshly ozen in OCT embedding
compound in liquid ni ogen cooled isopen ane and la e fixed wi h
me hanol ( o immunofluo escence). The IHC s ainings we e ca ied
ou on 4–6μm hick issue sec ions using he ollowing p ima y an i-
bodies: abbi an i- u in, (H-220), (San a C uz Bio echnology, Dallas,
TX), a an i-CD31 (BD Pha mingen, San Diego, CA) and a an i-F4/80
(Li e Technologies, Paisley, UK) ollowed by ho se adish pe oxidase
(HRP) o fluo escein-conjuga ed seconda y an ibodies. Hema oxylin
s aining was used as a coun e s ain. IF samples we e moun ed wi h
Vec ashield moun ing medium wi h DAPI (Vec o Labo a o ies,
Bu lingame, CA) and images we e aken wi h con ocal mic oscope (Ca l
Zeiss LSM 700). As a nega i e con ol, each s aining included sec ions
s ained wi hou p ima y an ibody.
4.6. Quan i a i e analysis o angiogenesis
Fo he analysis o e inal ascula u e, eyes we e enuclea ed, fixed
wi h 4% PFA and e inas dissec ed. Fla moun e inas we e blocked in
10% no mal goa se um o 2 h, incuba ed o e nigh wi h Isolec in GS-
IB
4
(1:200, In i ogen, Ca lsbad, CA) Re inas we e imaged ia con ocal
mic oscope (Ca l Zeiss LSM 700) and he a e o angiogenesis was de-
e mined du ing de elopmen (P6) and in he OIR model as p e iously
desc ibed (Conno e al., 2009; S ahl e al., 2010b). B iefly, e inas we e
imaged using con ocal mic oscopy (Ca l Zeiss LSM 700) wi h 10 × ob-
jec i e by ocusing on he p e e inal neo ascula u s and he unde -
lying supe ficial ascula plexus. A eas o ascula obli e a ion and
pa hological neo ascula iza ion (neo ascula u s) we e compu ed, in
pixels, and di ided by he o al e inal a ea using Adobe Pho oshop
CS3. The a e o de elopmen al angiogenesis a P6 was de e mined by
measu ing he diame e o ascula u e ia he op ic ne e o he ips o
he blood essels. Analysis o essel b anching and numbe o filopodia
sp ou s we e quan ified as p e iously desc ibed (Lobo e al., 2007).
Each poin whe e h ee capilla y segmen s me was coun ed as one
junc ion. B anch poin s om he capilla y plexus om wo a eas
(370 μm × 550 μm) pe e ina we e coun ed and esul s a e shown by
he a e age coun pe uni a ea. The numbe o filopodia p o usions
om he ip cells we e coun ed om he leng h o 500 μm o he
sp ou ing ascula on ( wo measu es pe e ina).
4.7. S a is ical analysis
S uden 's - es was conduc ed o no mally dis ibu ed da a and
nonpa ame ic Mann-Whi ney U es (G aphPad P ism 6.01 and IBM
SPSS s a is ics) o non-no mally dis ibu ed da a o es he s a is ical
significance o he esul s. P- alues less han 0.05 we e conside ed
s a is ically significan .
5. S a emen o au ho con ibu ions
M.V, T.J and H. U-J designed he esea ch. M.V and H.B. pe o med
he esea ch. M.V, H.B, T.J and H. U-J analyzed he da a. M.P, S.A and
Z.M.C p o ided LysMC e- u
(fl/fl)
mice o he s udy. M.V con ibu ed
he geno yped mice li e ma es. M.V, Z.M.C, H.B, T.J and H. U-J w o e
he manusc ip . M.V made he figu es. All au ho s e iewed he pape .
Conflic o in e es s a emen
The au ho s decla e no conflic o in e es .
Acknowledgemen s
We hank Ma ianne Ka lsbe g o excellen echnical assis ance. The
wo k was suppo ed by he Sig id Juselius Founda ion, he Academy o
Finland, Päi ikki and Saka i Sohlbe g Founda ion, Ins umen a ium
Resea ch Founda ion, Finnish Medical Founda ion, Pi kanmaa Hospi al
Dis ic Resea ch Founda ion and he Finnish Cul u al Founda ion,
Finnish Diabe ic Resea ch Founda ion, Finnish Eye Founda ion.
Re e ences
Abcouwe , S.F., 2012. Neu al inflamma ion and he mic oglial esponse in diabe ic e-
inopa hy. J. Ocul. Biol. Dis. In o 4, 25–33.
Amin, R., Puklin, J.E., F ank, R.N., 1994. G ow h ac o localiza ion in cho oidal neo-
ascula memb anes o age- ela ed macula degene a ion. In es . Oph halmol. Vis.
Sci. 35, 3178–3188.
Bai, Y., Liang, S., Yu, W., Zhao, M., Huang, L., Zhao, M., Li, X., 2014. Semapho in 3A
blocks he o ma ion o pa hologic cho oidal neo ascula iza ion induced by ans-
o ming g ow h ac o β. Mol. Vis. 20, 1258–1270.
Ba ke , H., Aal onen, M., Pan, P., Vähä upa, M., Kaipiainen, P., May, U., P ince, S.,
Uusi alo-Jä inen, H., Waheed, A., Pas o eko a, S., Sly, W.S., Pa kkila, S., Jä inen,
T.A., 2017. Role o ca bonic anhyd ases in skin wound healing. Exp. Mol. Med. 49,
e334.
Campochia o, P.A., 2015. Molecula pa hogenesis o e inal and cho oidal ascula dis-
eases. P og. Re in. Eye Res. 49, 67–81.
Checchin, D., Sennlaub, F., Le a asseu , E., Leduc, M., Chem ob, S., 2006. Po en ial ole
o mic oglia in e inal blood essel o ma ion. In es . Oph halmol. Vis. Sci. 47,
Fig. 2. Fu in deficiency in myeloid cells inhibi s e ascula iza ion a e in he e ina du ing he OIR model. Eyes we e ha es ed om heal hy and LysMC e- u (
fl/fl
) mice a P6
when he de elopmen o he supe ficial ascula plexus has occu ed and in he OIR model a P12 and P17. Re inas we e fixed wi h 4% PFA, fla moun ed and s ained wi h Alexa Fluo
conjuga ed Isolec in IB
4
. (A) The a e o ea ly angiogenesis was quan ified in e inal fla moun s by measu ing he leng h o ascula u e ia he op ic ne e. (B) The e was no diffe ence in
he leng h o angiogenic ascula u e o supe ficial ascula plexus be ween WT and LysMC e- u (
fl/fl
) e inas a P6. (C) The numbe o b anch poin s (whi e do s) in e inal ascula plexus
and filopodia p o usions (yellow do s) om ip cells we e coun ed om P6 e inas. The esul s a e exp essed as he numbe o b anch poin s pe uni a ea (D) and filopodia pe 500 μmo
ascula on (E). Bo h ou comes we e simila be ween geno ypes. WT n = 7 and KO n = 6. (F) Rep esen a i e images o e inas igh a e he hype oxic phase o OIR a P12. (G)
Quan i a i e analysis o he a ascula a eas (inside he whi e line) shows ha he ascula eg ession is simila in WT and LysMC e- u (
fl/fl
) animals. (WT n = 4, KO n = 7 e inas.) (H)
Rep esen a i e OIR model e inas o WT and LysMC e- u (
fl/fl
) mice a P17. (I) The a ascula a eas (yellow) and p e e inal pa hological neo ascula iza ion ( u s, whi e) we e quan ified.
(J) Re ascula iza ion a e o e ina is educed by 30% (p = 0.012) in he LysMC e- u (
fl/fl
) mice, while no diffe ences we e de ec ed in he pa hological e ascula iza ion, i.e. in he
p e e inal u s (K). (WT n = 26 and KO n = 19 e inas.) Scale ba ep esen s 1 mm in A, F, H and I and 200 μm in C. E o ba s ep esen SDs.
M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167
165
3595–3602.
Conno , K.M., K ah, N.M., Dennison, R.J., Ade man, C.M., Chen, J., Gue in, K.I., Sapieha,
P., S ahl, A., Wille , K.L., Smi h, L.E., 2009. Quan ifica ion o oxygen-induced e-
inopa hy in he mouse: a model o essel loss, essel eg ow h and pa hological
angiogenesis. Na . P o oc. 4, 1565–1573.
Co do a, Z.M., G onholm, A., Ky ola, V., Ta e ni i, V., Hamalainen, S., Ai omäki, S.,
Niininen, W., Jun ila, I., Ylipaa, A., Nyk e , M., Pesu, M., 2016. Myeloid cell ex-
p essed p op o ein con e ase FURIN a enua es inflamma ion. Onco a ge 23
7(34):54392–54404.
Cui, Y., Jean, F., Thomas, G., Ch is ian, J.L., 1998. BMP-4 is p o eoly ically ac i a ed by
u in and/o PC6 du ing e eb a e emb yonic de elopmen . EMBO J. 17,
4735–4743.
Da ies, M.H., Eubanks, J.P., Powe s, M.R., 2006. Mic oglia and mac ophages a e in-
c eased in esponse o ischemia-induced e inopa hy in he mouse e ina. Mol. Vis.
12, 467–477.
Dubois, C.M., Blanche e, F., Lap ise, M.H., Leduc, R., G ondin, F., Seidah, N.G., 2001.
E idence ha u in is an au hen ic ans o ming g ow h ac o -β1-con e ing en-
zyme. Am. J. Pa hol. 158, 305–316.
FANTOM Conso ium, he RIKEN PMI, CLST (DGT),Fo es , A.R., Kawaji, H., Rehli, M.,
Baillie, J.K., de Hoon, M.J., Habe le, V., Lassmann, T., Kulako skiy, I.V., Lizio, M.,
I oh, M., Ande sson, R., Mungall, C.J., Meehan, T.F., Schmeie , S., Be in, N.,
Jo gensen, M., Dimon , E., A ne , E., Schmidl, C., Schae e , U., Med ede a, Y.A.,
Plessy, C., Vi ezic, M., Se e in, J., Semple, C., Ishizu, Y., Young, R.S., F ancesca o,
M., Alam, I., Albanese, D., Al schule , G.M., A akawa, T., A che , J.A., A ne , P.,
Babina, M., Rennie, S., Balwie z, P.J., Beckhouse, A.G., P adhan-Bha , S., Blake, J.A.,
Blumen hal, A., Bodega, B., Bone i, A., B iggs, J., B ombache , F., Bu oughs, A.M.,
Cali ano, A., Cannis aci, C.V., Ca bajo, D., Chen, Y., Chie ici, M., Ciani, Y., Cle e s,
H.C., Dalla, E., Da is, C.A., De ma , M., Diehl, A.D., Dohi, T., D ablos, F., Edge, A.S.,
Edinge , M., Ekwall, K., Endoh, M., Enomo o, H., Fagiolini, M., Fai bai n, L., Fang,
H., Fa ach-Ca son, M.C., Faulkne , G.J., Fa o o , A.V., Fishe , M.E., F i h, M.C.,
Fuji a, R., Fukuda, S., Fu lanello, C., Fu ino, M., Fu usawa, J., Geij enbeek, T.B.,
Gibson, A.P., Ginge as, T., Goldowi z, D., Gough, J., Guhl, S., Gule , R., Gus incich,
S., Ha, T.J., Hamaguchi, M., Ha a, M., Ha be s, M., Ha shba ge , J., Hasegawa, A.,
Hasegawa, Y., Hashimo o, T., He lyn, M., Hi chens, K.J., Ho Sui, S.J., Ho mann, O.M.,
Hoo , I., Ho i, F., Huminiecki, L., Iida, K., Ikawa, T., Janko ic, B.R., Jia, H., Joshi, A.,
Ju man, G., Kaczkowski, B., Kai, C., Kaida, K., Kaiho, A., Kajiyama, K., Kanamo i-
Ka ayama, M., Kasiano , A.S., Kasukawa, T., Ka ayama, S., Ka o, S., Kawaguchi, S.,
Kawamo o, H., Kawamu a, Y.I., Kawashima, T., Kempfle, J.S., Kenna, T.J., Ke e, J.,
Khachigian, L.M., Ki amu a, T., Klinken, S.P., Knox, A.J., Kojima, M., Kojima, S.,
Kondo, N., Koseki, H., Koyasu, S., K ampi z, S., Kubosaki, A., Kwon, A.T., La os, J.F.,
Lee, W., Lenna sson, A., Li, K., Lilje, B., Lipo ich, L., Mackay-Sim, A., Manabe, R.,
Ma , J.C., Ma chand, B., Ma helie , A., Mejhe , N., Meyne , A., Mizuno, Y., de Lima
Mo ais, D.A., Mo ikawa, H., Mo imo o, M., Mo o, K., Mo akis, E., Mo ohashi, H.,
Mumme y, C.L., Mu a a, M., Nagao-Sa o, S., Nakachi, Y., Nakaha a, F., Nakamu a, T.,
Nakamu a, Y., Nakaza o, K., an Nimwegen, E., Ninomiya, N., Nishiyo i, H., Noma,
S., Noma, S., Noazaki, T., Ogishima, S., Ohku a, N., Ohimiya, H., Ohno, H., Ohshima,
M., Okada-Ha akeyama, M., Okazaki, Y., O lando, V., O chinniko , D.A., Pain, A.,
Passie , R., Pa ikakis, M., Pe sson, H., Piazza, S., P ende gas , J.G., Rackham, O.J.,
Ramilowski, J.A., Rashid, M., Ra asi, T., Rizzu, P., Roncado , M., Roy, S., Rye, M.B.,
Saijyo, E., Sajan ila, A., Saka, A., Sakaguchi, S., Sakai, M., Sa o, H., Sa i, S., Saxena,
A., Schneide , C., Schul es, E.A., Schulze-Tanzil, G.G., Schwegmann, A., Sengs ag, T.,
Sheng, G., Shimoji, H., Shimoni, Y., Shin, J.W., Simon, C., Sugiyama, D., Sugiyama,
T., Suzuki, M., Suzuki, N., Swoboda, R.K., Hoen, P.A., Tagami, M., Takahashi, N.,
Takai, J., Tanaka, H., Ta sukawa, H., Ta um, Z., Thompson, M., Toyodo, H., Toyoda,
T., Valen, E., an de We e ing, M., an den Be g, L.M., Ve ado, R., Vijayan, D.,
Vo on so , I.E., Wasse man, W.W., Wa anabe, S., Wells, C.A., Win e ingham, L.N.,
Wol e ang, E., Wood, E.J., Yamaguchi, Y., Yamamo o, M., Yoneda, M., Yoneku a, Y.,
Yoshida, S., Zabie owski, S.E., Zhang, P.G., Zhao, X., Zucchelli, S., Summe s, K.M.,
Suzuki, H., Daub, C.O., Kawai, J., Heu ink, P., Hide, W., F eeman, T.C., Lenha d, B.,
Bajic, V.B., Taylo , M.S., Makee , V.J., Sandelin, A., Hume, D.A., Ca ninci, P.,
Hayashizaki, Y., 2014. A p omo e -le el mammalian exp ession a las. Na u e 507,
462–470.
Fong, D.S., Aiello, L., Ga dne , T.W., King, G.L., Blankenship, G., Ca alle ano, J.D., Fe is
3 d, F.L., Klein, R., Ame ican Diabe es Associa ion, 2004. Re inopa hy in diabe es.
Diabe es Ca e 27 (Suppl. 1), S84–S87.
Gao, X., Wang, Y.S., Li, X.Q., Hou, H.Y., Su, J.B., Yao, L.B., Zhang, J., 2016. Mac ophages
p omo e asculogenesis o e inal neo ascula iza ion in an oxygen-induced e ino-
pa hy model in mice. Cell Tissue Res. 364, 599–610.
Jonas, J.B., Mones, J., Glace -Be na d, A., Coscas, G., 2017. Re inal ein occlusions. De .
Oph halmol. 58, 139–167.
Kanda, A., Noda, K., Sai o, W., Ishida, S., 2012. (P o) enin ecep o is associa ed wi h
angiogenic ac i i y in p oli e a i e diabe ic e inopa hy. Diabe ologia 55,
3104–3113.
Ka aoka, K., Nishiguchi, K.M., Kaneko, H., an Rooijen, N., Kachi, S., Te asaki, H., 2011.
The oles o i eal mac ophages and ci cula ing leukocy es in e inal neo ascula -
iza ion. In es . Oph halmol. Vis. Sci. 52, 1431–1438.
Kha ib, A.M., Lahlil, R., Scamuffa, N., Akimenko, M.A., E nes , S., Lom i, A., Lalou, C.,
Seidah, N.G., Villou eix, B.O., Cal o, F., Sieg ied, G., 2010. Zeb afish P oVEGF-C
exp ession, p o eoly ic p ocessing and inhibi o y effec o unp ocessed P oVEGF-C
du ing fin egene a ion. PLoS One 5, e11438.
Kim, W., Essalmani, R., Szumska, D., C eeme s, J.W., Roeb oek, A.J., D'O leans-Jus e, P.,
Bha acha ya, S., Seidah, N.G., P a , A., 2012. Loss o endo helial u in leads o
ca diac mal o ma ion and ea ly pos na al dea h. Mol. Cell. Biol. 32, 3382–3391.
Liyanage, S.E., Fan in, A., Villacampa, P., Lange, C.A., Den i, L., C is an e, E., Smi h, A.J.,
Ali, R.R., Luhmann, U.F., Bainb idge, J.W., Ruh be g, C., 2016. Myeloid-de i ed
ascula endo helial g ow h ac o and hypoxia-inducible ac o a e dispensable o
ocula neo ascula iza ion–b ie epo . A e ioscle . Th omb. Vasc. Biol. 36, 19–24.
Lobo , I.B., Rena d, R.A., Papadopoulos, N., Gale, N.W., Thu s on, G., Yancopoulos, G.D.,
Wiegand, S.J., 2007. Del a-like ligand 4 (Dll4) is induced by VEGF as a nega i e
egula o o angiogenic sp ou ing. P oc. Na l. Acad. Sci. U. S. A. 104, 3219–3224.
Lopez de Cicco, R., Wa son, J.C., Bassi, D.E., Li win, S., Klein-Szan o, A.J., 2004.
Simul aneous exp ession o u in and ascula endo helial g ow h ac o in human
o al ongue squamous cell ca cinoma p og ession. Clin. Cance Res. 10, 4480–4488.
Ma, J., E a d, S., Badiola, I., Sieg ied, G., Kha ib, A.M., 2017. Regula ion o he p o-
p o ein con e ases exp ession and ac i i y du ing egene a i e angiogenesis: ole o
hypoxia-inducible ac o (HIF). Eu . J. Cell Biol. 96, 457–468.
McColl, B.K., Paa onen, K., Ka nezis, T., Ha is, N.C., Da ydo a, N., Ro hacke , J., Nice,
E.C., Ha de , K.W., Rou ail, S., Hibbs, M.L., Roge s, P.A., Ali alo, K., S acke , S.A.,
Achen, M.G., 2007. P op o ein con e ases p omo e p ocessing o VEGF-D, a c i ical
s ep o binding he angiogenic ecep o VEGFR-2. FASEB J. 21, 1088–1098.
McMahon, S., G ondin, F., McDonald, P.P., Richa d, D.E., Dubois, C.M., 2005. Hypoxia-
enhanced exp ession o he p op o ein con e ase u in is media ed by hypoxia-in-
ducible ac o -1: impac on he bioac i a ion o p op o eins. J. Biol. Chem. 280,
6561–6569.
Mi amu a, Y., Ha ada, C., Ha ada, T., 2005. Role o cy okines and ophic ac o s in he
pa hogenesis o diabe ic e inopa hy. Cu . Diabe es Re . 1, 73–81.
Oksanen, A., Ai omäki, S., Janko ic, D., O u ay, Z., Pulkkinen, K., Hamalainen, S.,
Rokka, A., Co hals, G.L., Wa o d, W.T., Jun ila, I., O'Shea, J.J., Pesu, M., 2014.
P op o ein con e ase FURIN cons ain Th2 diffe en ia ion and is c i ical o hos
esis ance agains Toxoplasma gondii. J. Immunol. 193, 5470–5479.
Pesu, M., Wa o d, W.T., Wei, L., Xu, L., Fuss, I., S obe , W., Ande sson, J., She ach,
E.M., Quezado, M., Bouladoux, N., Roeb oek, A., Belkaid, Y., C eeme s, J., O'Shea,
J.J., 2008. T-cell-exp essed p op o ein con e ase u in is essen ial o main enance
o pe iphe al immune ole ance. Na u e 455, 246–250.
Reynolds, J.D., 2014. Insigh s in ROP. Am. O hop . J. 64, 43–53.
Roeb oek, A.J., Umans, L., Pauli, I.G., Robe son, E.J., an Leu en, F., Van de Ven, W.J.,
Cons am, D.B., 1998. Failu e o en al closu e and axial o a ion in emb yos lacking
he p op o ein con e ase Fu in. De elopmen 125, 4863–4876.
Scamuffa, N., Cal o, F., Ch e ien, M., Seidah, N.G., Kha ib, A.M., 2006. P op o ein con-
e ases: lessons om knockou s. FASEB J. 20, 1954–1963.
Siege , S., Cabuy, E., Sche , B.G., Kohle , H., Panda, S., Le, Y.Z., Fehling, H.J., Gaida zis,
D., S adle , M.B., Roska, B., 2012. T ansc ip ional code and disease map o adul
e inal cell ypes. Na . Neu osci. 15 487–495, S1-2.
Sieg ied, G., Basak, A., C omlish, J.A., Benjanne , S., Ma cinkiewicz, J., Ch e ien, M.,
Seidah, N.G., Kha ib, A.M., 2003a. The sec e o y p op o ein con e ases u in, PC5,
and PC7 ac i a e VEGF-C o induce umo igenesis. J. Clin. In es . 111, 1723–1732.
Sieg ied, G., Kha ib, A.M., Benjanne , S., Ch e ien, M., Seidah, N.G., 2003b. The p o-
eoly ic p ocessing o p o-pla ele -de i ed g ow h ac o -A a RRKR(86) by membe s
o he p op o ein con e ase amily is unc ionally co ela ed o pla ele -de i ed
g ow h ac o -A-induced unc ions and umo igenici y. Cance Res. 63, 1458–1463.
Sieg ied, G., Basak, A., P iche -Pejic, W., Scamuffa, N., Ma, L., Benjanne , S., Veino ,
J.P., Cal o, F., Seidah, N., Kha ib, A.M., 2005. Regula ion o he s epwise p o eoly ic
clea age and sec e ion o PDGF-B by he p op o ein con e ases. Oncogene 24,
6925–6935.
Sil es i, L., Pagani, A., Camaschella, C., 2008. Fu in-media ed elease o soluble hemo-
ju elin: a new link be ween hypoxia and i on homeos asis. Blood 111, 924–931.
Smi h, L.E., Wesolowski, E., McLellan, A., Kos yk, S.K., D'Ama o, R., Sulli an, R.,
D'Amo e, P.A., 1994. Oxygen-induced e inopa hy in he mouse. In es . Oph halmol.
Vis. Sci. 35, 101–111.
S ahl, A., Chen, J., Sapieha, P., Seawa d, M.R., K ah, N.M., Dennison, R.J., Fa azza, T.,
Buche , F., Lo q is , C., Ong, H., Hells om, A., Chem ob, S., Akula, J.D., Smi h, L.E.,
2010a. Pos na al weigh gain modifies se e i y and unc ional ou come o oxygen-
induced p oli e a i e e inopa hy. Am. J. Pa hol. 177, 2715–2723.
S ahl, A., Conno , K.M., Sapieha, P., Chen, J., Dennison, R.J., K ah, N.M., Seawa d, M.R.,
Wille , K.L., Ade man, C.M., Gue in, K.I., Hua, J., Lo q is , C., Hells om, A., Smi h,
L.E., 2010b. The mouse e ina as an angiogenesis model. In es . Oph halmol. Vis. Sci.
51, 2813–2826.
Tammela, T., Za kada, G., Nu mi, H., Jakobsson, L., Heinolainen, K., T o ogo , D.,
Zheng, W., F anco, C.A., Mu omaki, A., A anda, E., Miu a, N., Yla-He uala, S.,
F u ige , M., Makinen, T., Eichmann, A., Polla d, J.W., Ge ha d , H., Ali alo, K.,
2011. VEGFR-3 con ols ip o s alk con e sion a essel usion si es by ein o cing
No ch signalling. Na . Cell Biol. 13, 1202–1213.
Tsu sumi, C., Sonoda, K.H., Egashi a, K., Qiao, H., Hisa omi, T., Nakao, S., Ishibashi, M.,
Cha o, I.F., Sakamo o, T., Mu a a, T., Ishibashi, T., 2003. The c i ical ole o ocula -
infil a ing mac ophages in he de elopmen o cho oidal neo ascula iza ion.
J. Leukoc. Biol. 74, 25–32.
Tu peinen, H., O u ay, Z., Pesu, M., 2013. Gene ics o he fi s se en p op o ein con-
e ase enzymes in heal h and disease. Cu . Genomics 14, 453–467.
Uusi alo-Jä inen, H., Ku okawa, T., Muelle , B.M., And ade-Go don, P., F iedlande , M.,
Ru , W., 2007. Role o p o ease ac i a ed ecep o 1 and 2 signaling in hypoxia-
induced angiogenesis. A e ioscle . Th omb. Vasc. Biol. 27, 1456–1462.
Vähä upa, M., Ai omäki, S., Ma inez Co do a, Z., May, U., P ince, S., Uusi alo-Jä inen,
H., Jä inen, T.A., Pesu, M., 2016a. T-cell-exp essed p op o ein con e ase FURIN
inhibi s DMBA/TPA-induced skin cance de elopmen . Oncoimmunology 5,
e1245266.
Vähä upa, M., P ince, S., Va aja, S., Me imo, T., Ka aja, M., Kinnunen, K., Ma jomaki, V.,
Uusi alo, H., Koma su, M., Jä inen, T.A., Uusi alo-Jä inen, H., 2016b. Lack o - as
leads o inc eased ascula pe meabili y in ischemic e inopa hy. In es . Oph halmol.
Vis. Sci. 57, 4898–4909.
Ven u a, E., Welle , M., Bu gha d , I., 2017. Cu ing edge: ERK1 media es he au oc ine
posi i e eedback loop o TGFβand u in in glioma-ini ia ing cells. J. Immunol. 198,
4569–4574.
M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167
166
Wang, X., Ma, W., Han, S., Meng, Z., Zhao, L., Yin, Y., Wang, Y., Li, J., 2017. TGFβ
pa icipa es cho oid neo ascula iza ion h ough Smad2/3-VEGF/TNF-αsignaling in
mice wi h Lase -induced we age- ela ed macula degene a ion. Sci. Rep. 7
9672–017-10124-4.
Yingchuan, F., Chun ao, L., Hui, C., Jianbin, H., 2010. Inc eased exp ession o TGF-β1 and
Smad 4 on oxygen-induced e inopa hy in neona al mice. Ad . Exp. Med. Biol. 664,
71–77.
Zhou, Y., Yoshida, S., Nakao, S., Yoshimu a, T., Kobayashi, Y., Nakama, T., Kubo, Y.,
Miyawaki, K., Yamaguchi, M., Ishikawa, K., Oshima, Y., Akashi, K., Ishibashi, T.,
2015. M2 mac ophages enhance pa hological neo ascula iza ion in he mouse model
o oxygen-induced e inopa hy. In es . Oph halmol. Vis. Sci. 56, 4767–4777.
M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167
167