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D
Lack o R-Ras Leads o Inc eased Vascula Pe meabili y in
Ischemic Re inopa hy
Ma ia V¨ah¨a upa,
1,2
S ua P ince,
2
Su i Va aja,
1
Teija Me imo,
1
Ma ko Ka aja,
3
Ka i Kinnunen,
4
Va pu Ma jom¨aki,
5
Hannu Uusi alo,
1,3
Masanobu Koma su,
6
Te o A.H. J¨a inen,
2,7
and Hannele Uusi alo–J¨a inen
1,3
1
Depa men o Oph halmology, Uni e si y o Tampe e, Tampe e, Finland
2
Depa men o Ana omy, Uni e si y o Tampe e, Tampe e, Finland
3
Eye Cen e, Tampe e Uni e si y Hospi al, Tampe e, Finland
4
Depa men o Oph halmology, Kuopio Uni e si y Hospi al, Kuopio, Finland
5
Depa men o Biological and En i onmen al Science/Nanoscience Cen e , Uni e si y o Jy ¨askyl¨a, Jy ¨askyl¨a, Finland
6
San o d Bu nham P ebys Medical Disco e y Ins i u e a Lake Nona, O lando, Flo ida, Uni ed S a es
7
Depa men o Musculoskele al Diso de s, Tampe e Uni e si y Hospi al, Tampe e, Finland
Co espondence: Hannele Uusi alo-
J¨a inen, Depa men o Oph hal-
mology, School o Medicine, 33014
Uni e si y o Tampe e, Finland;
llhauus@u a. i
Submi ed: Janua y 25, 2016
Accep ed: July 14, 2016
Ci a ion: Va¨h¨a upa M, P ince S, Va aja
S, e al. Lack o R-Ras leads o
inc eased ascula pe meabili y in
ischemic e inopa hy.
In es Oph hal-
molVisSci
. 2016;57:4898–4909.
DOI:10.1167/io s.16-19212
P
URPOSE
.
The ole o R-Ras in e inal angiogenesis and ascula pe meabili y was e alua ed in
an oxygen-induced e inopa hy (OIR) model using R-Ras knockou (KO) mice and in human
diabe ic neo ascula memb anes.
M
ETHODS
.
Mice de icien o R-Ras and hei wild- ype (WT) li e ma es we e subjec ed o 75%
oxygen om pos na al day 7 (P7) o P12 and hen e u ned o oom ai . A P17 e inal
ascula iza ion was examined om whole moun s, and e inal ascula pe meabili y was
s udied using Miles assay. Real- ime RT-PCR, Wes e n blo ing, and immunohis ochemis y
we e used o assess he exp ession o R-Ras in e ina du ing de elopmen o in he OIR
model. The deg ee o pe icy e co e age and ascula endo helial (VE)-cadhe in exp ession on
WT and R-Ras KO e inal blood essels was quan i ied using con ocal mic oscopy. The
co ela ion o R-Ras wi h ascula endo helial g ow h ac o ecep o 2 (VEGFR2) and human
se um albumin on human p oli e a i e diabe ic e inopa hy memb anes was assessed using
immunohis ochemis y.
R
ESULTS
.
In e ina, R-Ras exp ession was mos ly es ic ed o he ascula u e. Re inal essels in
he R-Ras KO mice we e signi ican ly mo e pe meable han WT con ols in he OIR model. A
signi ican educ ion in he di ec physical con ac be ween pe icy es and blood essel
endo helium as well as educed VE-cadhe in immunos aining was ound in R-Ras–de icien
mice. In human p oli e a i e diabe ic e inopa hy neo ascula memb anes, R-Ras exp ession
nega i ely co ela ed wi h inc eased ascula leakage and exp ession o VEGFR2, a ma ke o
blood essel imma u i y.
C
ONCLUSIONS
.
Ou esul s sugges ha R-Ras has a ole in con olling e inal essel ma u a ion
and s abiliza ion in ischemic e inopa hy and p o ides a po en ial a ge o pha macologic
manipula ion o ea diabe ic e inopa hy.
Keywo ds: e ina, diabe ic e inopa hy, neo ascula iza ion, e inal ischemia
iabe ic macula edema (DME) and p oli e a i e diabe ic
e inopa hy (PDR) a e he wo o ms o diabe ic e inop- a hy
causing blindness. Vascula endo helial g ow h ac o
(VEGF) plays a c ucial ole in hese diseases by causing ascula
leakage and pa hologic neo ascula iza ion.
1–3
An i-VEGF d ugs
a e ega ded as he mains ay o ea men o pa ien s a ec ed
by DME. Howe e , VEGF inhibi ion ails o p o ide su icien
e icacy in all DME cases; only hal o pa ien s a ain signi ican
imp o emen in isual acui y.
4
An i-VEGF he apy is also
accompanied by a e, bu se e e, ocula and sys emic side
e ec s. In PDR, an i-VEGF may be associa ed wi h de elopmen
o ac ional e inal de achmen
5,6
Fu he mo e, in a i eally
adminis e ed an i-VEGF agen s may cause sus ained sys emic
VEGF inhibi ion
7,8
This is why hey a e ela i ely con aindi-
ca ed in p egnan women
9
and pa ien s wi h ca dio ascula
insu iciency.
10,11
Thus, al e na i e he apies a e needed o
mo e e ec i e and sa e ea men o diabe ic e inopa hy.
12,13
R-Ras is a small GTPase o he Ras amily o oncogenes.
14
Despi e he close s uc u al simila i y o o he membe s o he
Ras amily, he unc ion o R-Ras is dis inc om o he Ras
p o eins.
15
Whe eas all o he membe s o he Ras amily may
cause malignan ans o ma ion, R-Ras has e y li le o no
ans o ming ac i i y.
16
Fu he mo e, he opposing unc ions o
R-Ras and H-Ras ex end o cell–ex acellula ma ix (ECM)
adhesion,
17
cell di e en ia ion,
18
and signaling.
19
These oppos-
ing unc ions ha e led o he sugges ion ha he balance
be ween R-Ras and o he Ras membe s ac s as a swi ch ha
con ols p oli e a ion and in asion e sus quiescence in
cells.
15,20,21
Recen ly, ac i e Ras signaling has been implica ed as a key
d i e o pa hologic angiogenesis in neo ascula diseases o he
io s.a ojou nals.o g
j
ISSN: 1552-5783
4898
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Lack o R-Ras and Vascula Pe meabili y in Re inopa hy IOVS jSep embe 2016 jVol. 57 jNo. 11 j4899
e ina.
22
Con e sely, exac ly he opposi e ole has ecen ly
been shown o R-Ras in he egula ion o angiogenesis.
20
R-Ras
unc ion has been shown o be essen ial o he es ablishmen
o ma u e and unc ional blood essels in umo s by
s abiliza ion o imma u e, leaky essels, enhancing pe usion
and ul ima ely educing plasma leakage.
15,21
Based on hese
unc ions, R-Ras has been classi ied as an an iangiogenic
molecule. Howe e , R-Ras is unc ionally di e en om classic
an iangiogenic agen s as i does no induce endo helial cell
apop osis as do o he an iangiogenic molecules, bu ac ually
p omo es endo helial cell su i al.
20
Thus, R-Ras could p o ide
a po en ial agonis ic he apeu ic a ge o ea men o a
a ie y o e inal diseases associa ed wi h ischemia, neo ascu-
la iza ion, and ascula leakage. In he p esen s udy, he ole o
R-Ras in he e ina was s udied by analyzing exp ession o R-Ras
in he e ina and by analyzing he e ec o R-Ras de iciency on
he s uc u al and unc ional in eg i y o e inal ascula u e
du ing de elopmen and ischemic e inopa hy using an
oxygen-induced e inopa hy model (OIR). To add ess he ole
o R-Ras in PDR, associa ion o i s exp ession wi h ascula
imma u i y and leakiness was examined in human e inal
neo ascula memb anes.
METHODS
Mice
Fo he expe imen s, wild- ype (WT) C57BL/6 mice om
Ha lan Sp ague Dawley (Indianapolis, IN, USA) and homozy-
gous R-Ras knockou (KO) mice we e used. R-Ras KO mice
wi h an inse ion dele ing R-Ras exp ession be ween exons 4
and 5 o
R as
on ch omosome 7 (R-Ras KO mice) ha e been
desc ibed p e iously.
20
Be o e any expe imen s, R-Ras he e o-
zygous mice we e backc ossed eigh imes wi h he C57BL/6
s ain o ob ain homozygous KO and WT mice in he same
gene ic backg ound. The mice we e 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 Vision Resea ch in acco dance
wi h p o ocols app o ed by he Na ional Animal E hics
Commi ee o Finland.
Oxygen-Induced Re inopa hy Model
The expe imen s on he OIR model we e ca ied ou as
desc ibed in de ail p e iously.
23,24
B ie ly, neona al mice a
pos na al day 7 (P7) we e exposed o 75% oxygen o 5 days. A
P12, hey we e e u ned o no mal oom ai .
23
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 a ec ou come in he OIR model,
25
only he
pups weighing be ween 6.3 and 7.5 g a P17 we e included in
he s udy.
Immunohis ochemis y (IHC) and Isolec in GS-IB
4
S aining
Fo he analysis o e inal ascula u e, eyes we e enuclea ed,
ixed wi h 4% pa a o maldehyde (PFA), and e inas dissec ed.
Fla -moun e inas we e blocked in 20% no mal goa and 20%
e al bo ine se ums o 2 hou s, incuba ed o e nigh Isolec in
(Isolec in GS-IB
4
, 1:200; In i ogen, Ca lsbad, CA, USA) and
wi h an i-NG2 chond oi in sul a e p o eoglycan an ibody (1:80;
Millipo e, Bille ica, MA), ollowed by Alexa Fluo -conjuga ed
seconda y an ibody. Re inas we e imaged ia con ocal mic o-
scope (LSM 700; Ca l Zeiss, Obe kochen, Ge many) and he
a e o angiogenesis was de e mined du ing de elopmen (P0–
P6) and in he OIR model as p e iously desc ibed.
24
B ie ly,
e inas we e imaged using con ocal mic oscopy (Ca l Zeiss LSM
700) wi h 5
3
objec i e. By ocusing jus abo e he inne
limi ing memb ane o he e ina, he p e e inal neo ascula
u s we e eadily dis inguished om he unde lying supe icial
ascula plexus. A eas o ascula obli e a ion and pa hologic
neo ascula iza ion (meaning neo ascula u s) we e quan i-
ied om hese images using image edi ing so wa e (Adobe
Pho oshop CS3; Adobe Sys ems, Inc., San Jose, CA, USA). The
a e o de elopmen al angiogenesis a P0 o P6 was de e mined
by measu ing he leng h o ascula u e om he op ic ne e o
he ips o he blood essels. Fou measu emen s pe e ina
we e aken and an a e age was calcula ed.
Fo immunohis ochemis y, he eyes we e ixed wi h 4%
PFA and embedded in pa a in o immedia ely ozen in
o ni hine ca bamoyl ans e ase embedding compound in iso-
pen ane cooled wi h liquid ni ogen and la e ixed wi h
ace one. The IHC was ca ied ou on 4- o 6-lm- hick issue
sec ions using he ollowing p ima y an ibodies: abbi an i–R-
Ras (1:50) and abbi an i-VEGFR2 (1:100; bo h Cell Signaling
Technology, Dan e s, MA, USA), abbi an i–R-Ras (1:750) and
mouse an i-VEGFR2 (1:200, clone sc.6251; bo h San a C uz
Bio echnology, Dallas, TX, USA), a an i-mouse CD31 (1:50;
BD Pha mingen, San Diego, CA, USA), guinea pig an i-NG2
an ibody (1:100, gi om William S allcup),
26
abbi an i-
Syndecan (1:500) and abbi an i-human ascula endo helial
(VE)-cadhe in (1:500; bo h om Abcam, Camb idge, UK), and
abbi an i-human se um albumin (1:100; Li espan Biosciences,
Sea le, WA, USA), ollowed by ho se adish pe oxidase (HRP)
o luo escein-conjuga ed seconda y an ibodies. Hema oxylin
s aining was used as a coun e s ain. Samples we e moun ed
wi h Vec ashield moun ing medium wi h 4
0
,6-diamidino-2-
phenylindole
(
Vec o Labo a o ies) and analyzed ia con ocal
mic oscope. Each s aining expe imen included sec ions
s ained wi hou p ima y an ibody as nega i e con ols.
Pe cen age o R-Ras posi i e cells in p e e inal neo essels
we e quan i ied manually by using he coun ool in Adobe
Pho oshop (Adobe Sys ems, Inc.) (
n
¼
6 mice, 4–6 sec ions/
e ina).
Quan i ica ion o Pe icy e Co e age and VE-
Cadhe in Colocaliza ion Wi h CD31
Pe icy e co e age o he blood essels was quan i ied om WT
and R-Ras KO P17 OIR model whole-moun e inas. Samples
we e imaged using con ocal mic oscopy (Ca l Zeiss LSM 700)
wi h a 63
3
objec i e lens, and 3D pic u es we e made om Z-
s acks. Pic u es selec ed o he analysis we e andomly aken
om Isolec in IB
4
and NG2-s ained la -moun s om he mos
supe icial ascula plexus a he ips o he blood essels nex
o a ascula a ea. This is a egion o e ina whe e he blood
essels a e g owing owa d he op ic ne e. Di ec con ac
be ween pe icy es (NG2) and endo helial cells (Isolec in B
4
)
we e quan i ied by colocaliza ion analysis wi h BioImageXD
(p o ided in he public domain a h p://www.bioimagexd.
o g).
27
The pe cen age o EC a ea colocalized wi h pe icy e
a ea was quan i ied in each image, and he esul exp essed as a
mean o wo di e en images o each e ina. The in ensi y o
VE-cadhe in s aining and colocaliza ion o VE-cadhe in and
CD31 was quan i ied om ozen sec ions using BioImage XD.
Real-Time Quan i a i e PCR (qPCR)
Eyes we e collec ed a P0, P4, P7, P12, P17, and P22 and
placed immedia ely in cold RNA s abiliza ion eagen (RNA-
la e ; Sigma-Ald ich Co p., S . Louis, MO, USA) o he
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Lack o R-Ras and Vascula Pe meabili y in Re inopa hy IOVS jSep embe 2016 jVol. 57 jNo. 11 j4900
du a ion o dissec ion o he e inas. RNA was ex ac ed using
RNA isola ion ki (RNeasy Mini Ki ; Qiagen, Hilden, Ge many)
ollowing he manu ac u e ’s ins uc ions. The in eg i y o
ex ac ed RNA was e i ied on o maldehyde aga ose gel.
Re e se ansc ip ion o RNA was ca ied ou using cDNA
syn hesis ki (Maxima Fi s S and cDNA Syn hesis Ki ;
The mo Fishe Scien i ic, Bos on, MA, USA), acco ding o
he manu ac u e ’s ins uc ions. Real- ime qPCR was done
using he PCR dye (SYBR G een; The mo Fishe Scien i ic)
me hod acco ding o he manu ac u e ’s ecommenda ions
and pe o med in whi e 96-well pla es (Mul iply PCR pla es;
Sa s ed , Nu¨mb ech , Ge many). The luo escence signal was
de ec ed using a sequence de ec ion sys em (ABI P ism 7000
Sequence De ec ion Sys em 1.2; Applied Biosys ems, Ca ls-
bad, CA, USA). The ollowing in on-spanning p ime s
speci ic o exon 3 and 4 o
R as
we e used: 5
0
-ACAGGCA
GAGTTTCAATGAG-30( o wa d), 50-GTTCTCCAGATCTGCC
TTG-3
0
( e e se).
Ppia
was used as an endogenous con ol
gene (50-CACCGTGTTCTTCGACATC-30and 50-ATTCTGTGA
AAGGAGGAACC-3
0
), allowing he compa ison o samples.
20
Two o h ee eplica es we e analyzed o each sample, and
he esul s we e exp essed as a mean o each sample.
Nega i e, no empla e (NTC) and no e e se ansc ip ase (no
RT), con ols we e included in e e y qPCR analysis. A mel ing
cu e analysis was pe o med o check o unspeci ic PCR
p oduc s (which didn’ occu ). Reac ion e iciencies o bo h
p ime pai s we e es ed wi h s anda d cu e analysis in
di e en ime poin s. Due o simila e iciencies o bo h
p ime pai s, he di e ences in gene exp ession le els we e
de e mined by he compa a i e C me hod (DDC me hod).
28
Vascula Pe meabili y Assay
Vascula pe meabili y was quan i ied by Miles assay in e inas
as desc ibed p e iously.
29
B ie ly, he mice we e injec ed wi h
2% E ans Blue ([EB], 150 lL/20g; Sigma-Ald ich Co p.)
in ape i oneally, and he ci cula ion ime was 24 h o P16
o 1 hou o P17 injec ed mice. Phospha e-bu e ed saline–
injec ed mice we e used as a nega i e con ol. Animals we e
eu hanized a P17, and blood was collec ed om he le
en icle. Re inas we e dissec ed and weighed. Blood was
cen i uged a 3,550
g
o 15 minu es, and plasma was dilu ed
1:100 and 1:1000 in N, N-dime hyl o mamide (Sigma-Ald ich
Co p.). All samples we e incuba ed in a shake (100 pm) in
200 lL N, N-dime hyl o mamide o e nigh a 78
8
C. Re inas
we e cen i uged a 17,000
g
o 45 minu es a 4
8
C, and he
supe na an s we e collec ed. The EB abso bance o each
sample was measu ed a 620 nm by spec opho ome e . EB
concen a ion in he e ina was p opo ioned and no malized
by he weigh o he e ina and he amoun o EB concen a ion
in he plasma.
30
Wes e n Blo ing
Re inas we e lysed in 10 lL o cold RIPA bu e pe 1 mg o
issue wi h added p o ease inhibi o (c
O
mple e; Roche, Basel,
Swi ze land) and phospha ase inhibi o (Hal ; The mo Fishe
Scien i ic).
The issues we e homogenized using CK14 beads and a
issue homogenize (P ecellys; Be in Technologies, Mon igny le-
B e onneux, F ance). F om each sample, 40
lg
o p o ein was
loaded pe well o a 4-12% g adien gel (NuPAGE; In i ogen,
Ca lsbad, CA, USA) and elec oblo ed on poly inylidene
luo ide memb anes (Immun-Blo; Bio-Rad Labo a o ies, He cu-
les, Cali o nia). Fo de ec ion o speci ic p o eins by immuno-
blo ing, he ollowing p ima y an ibodies we e used: abbi
an i–R-Ras (1:500; Cell Signaling Technology), goa an i-GAPDH
(1:500: AbCam) and ho se an i-mouse IgG (1:2000; Cell
Signaling Technology). P ima y an ibodies we e de ec ed by
HRP-coupled an ibodies. Wes e n blo images we e cap u ed ia
so wa e (ImageQuan ; GE Heal hca e, Chal on S . Giles, UK)
and quan i ied by densi ome y using Adobe Pho oshop CS3
so wa e, whe e GAPDH was used o no malize o p o ein
loading. A calib a o con ol sample was included in e e y
memb ane o enable sample compa ison be ween di e en
memb anes.
Re inal and Neo ascula Samples F om Human
Pa ien s
P e e inal neo ascula memb anes we e ob ained om eigh
ype I diabe ic pa ien s who we e unde going pa s plana
i ec omy o he ea men o PDR. All pa ien s we e
Caucasians, and al oge he he e we e i e emales and h ee
males. A he ime o pa s plana, i ec omy pa ien s’ mean
age was 33 yea s ( ange, 27–56 yea s) and mean du a ion o
diabe es was 24 yea s ( ange, 16–32 yea s). No mal human
e inas we e ob ained om pa ien s whose eyes we e
enuclea ed due o cho oidal melanoma. The p o ocol o
collec ing human issue samples was app o ed by he
ins i u ional e iew boa ds o he Pi kanmaa Hospi al
Dis ic and he Kuopio Uni e si y Hospi al. The s udy was
conduc ed in acco dance wi h he Decla a ion o Helsinki.
All pa ien s ga e w i en in o med consen . Du ing i ec o-
my, he ib o ascula memb anes we e isola ed, g asped
wi h i eous o ceps, and pulled ou h ough a scle o omy.
The sample was immedia ely ixed wi h 4% o maldehyde o
3 h, ans e ed o 70% e hanol, embedded in pa a in, and
p ocessed o immunohis ochemis y. The numbe o R-Ras–
posi i e neo essels was calcula ed om CD31 and R-Ras–
double-s ained issue sec ions, and co ela ion be ween R-
Ras and VEGFR2 was quan i ied om R-Ras and VEGFR2
double-s ained sec ions. Immunohis ochemis y agains hu-
man se um albumin (HSA) was done o quan i y he amoun
o ascula leakage ou side he neo essels. Quan i ica ion
was done using IHC P o ile plugin in ImageJ so wa e
(h p://imagej.nih.go /ij/; p o ided in he public domain by
Na ional Ins i u es o Heal h, Be hesda, MD, USA). The a ea
o posi i e s aining o HSA was compa ed o he o al
analyzed a ea, and co ela ion analysis be ween HSA and
pe cen age o R-Ras–posi i e neo essels was pe o med o
each sample.
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 using s a is ical
so wa e (G aphPad P ism 6.01; G aphPad So wa e, San
Diego, CA, USA, and IBM SPSS S a is ics; IBM, Amonk, NY,
USA) o nonno mally dis ibu ed da a o es he s a is ical
signi icance o he esul s. Spea man’s ank co ela ion es was
conduc ed o es co ela ion be ween wo a iables.
P
alues
less han 0.05 we e conside ed s a is ically signi ican .
RESULTS
R-Ras Exp ession Peaks Du ing Re inal
De elopmen al Angiogenesis Bu Does No
In luence he Ra e o De elopmen al Angiogenesis
R-R
as KO mice a e e ile and show no ob ious mo phologic
abno mali ies,
20,31
and hei eyes appea no mal upon his o-
logic examina ion. To add ess he ole o R-Ras on e inal
angiogenesis in i o, he exp ession p o ile o R-Ras in he
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Lack o R-Ras and Vascula Pe meabili y in Re inopa hy IOVS jSep embe 2016 jVol. 57 jNo. 11 j4901
F
IGURE
1.
Hypoxia-d i en R-Ras exp ession in he OIR model. Hypoxia-induced angiogenesis in e ina was s udied wi h he OIR model. Re inas
we e ha es ed immedia ely a e he exposu e o hype oxia (P12) and a e hypoxia-d i en pa hologic angiogenesis has eached i s maximum a
P17. Re inas om no mal mice we e ha es ed a co esponding ime poin s. Re inas we e subjec ed o ei he quan i a i e mRNA ( eal- ime qPCR)
analysis using SYBR G een me hod o p o ein (Wes e n blo ing) analysis. Fo immunoblo ing, e inal p o ein supe na an s we e elec opho esed
on g adien gels, s anda d Wes e n blo ing was ca ied ou wi h R-Ras–speci ic an ibody, and GAPDH de ec ion was used as a loading con ol. (
A
)
The g aph ep esen s old changes
(2
-
DDC
me hod) in he
R as
mRNA exp ession le el ela i e o he
R as
mRNA exp ession le el o no mal mice a
P12. The le el o
R as
mRNA exp ession be ween no mal and OIR model e inas is equal a P12, whe eas a P17 hypoxia leads o 2.5- old
up egula ion o
R as
mRNA (
P
¼
0.016,
*
nonpa ame ic Mann-Whi ney
U
es ).
E o ba s
ep esen he minimum and maximum o he old
change. (P12, P17 OIR:
n
¼
4; P17, P12 OIR:
n
¼
5.) (
B
,
C
) The le el o R-Ras p o ein exp ession was quan i ied by densi ome ic analysis o
immunoblo ed p o ein. R-Ras p o ein le el a P17 shows a 7.5- old inc ease (
P
¼
0.016) du ing hypoxia-induced angiogenesis.
E o ba s
ep esen
6
95% con idence in e als. (P12:
n
¼
6; P12 OIR:
n
¼
7; P17:
n
¼
5; P17:
n
¼
5.) The esul s a e analyzed wi h nonpa ame ic Mann-Whi ney
U
es .
The samples p esen ed abo e we e un on he same gel. Rep esen a i e samples we e c opped and p esen ed side-by-side.
de eloping mouse e ina as well as he de elopmen al a e o
angiogenesis in WT and R-Ras KO mice we e cha ac e ized.
To assess he empo al exp ession pa e n o R-Ras du ing
de elopmen al e inal angiogenesis, qPCR and Wes e n blo
analysis we e used. Quan i a i e PCR demons a ed ha he
R as
mRNA exp ession inc eased om P0 onwa d, peaked a
P17, and s a ed o decline om P17 un il P22. Wes e n blo
analysis demons a ed a R-Ras p o ein exp ession p o ile
simila o he mRNA p o ile: an inc ease in exp ession om
P0 un il P17 and a decline om P17 o P22 (Supplemen a y
Fig. S1). The inc ease in
R as
mRNA and p o ein le els om
P0 o P17 co ela es wi h he de elopmen o e inal
ascula plexuses.
32
The R-Ras le els peaked a P17, when
all h ee ascula plexuses a e o med and ascula densi y
peaks in he e ina
33
(Supplemen a y Fig. S1). The indings
a e consis en wi h he no ion ha R-Ras appea s in he
blood essels du ing hei di e en ia ion and is mos
s ongly exp essed in ully di e en ia ed, quiescen blood
essels.
20
The educ ion in
R as
mRNA and p o ein
exp ession le els om P17 o P22 can be explained by he
emodeling o e inal ascula u e (including ascula p un-
ing), he ou come o which is dec eased endo helial cell
densi y a P28.
34
Consis en wi h his inding, he esul s
show a simila dec ease in he in ensi y o Isolec in IB
4
s aining om P12 onwa d (Supplemen a y Fig. S2). No R-Ras
p o ein o mRNA exp ession was de ec ed in he e inas
collec ed om R-Ras KO mice.
To explo e whe he he inc ease in R-Ras exp ession le el
seen du ing e inal de elopmen in luences he a e o
supe icial ascula plexus o ma ion (de elopmen al angio-
genesis), e inas om P0, P2, P4, and P6 WT and R-Ras KO
mice we e analyzed. In P0, P2, P4, and P6 neona al mice, he
diame e o e inal supe icial ascula plexuses we e simila
bo h in R-Ras KO and WT mice, indica ing ha R-Ras
exp ession is no necessa y o in i o angiogenesis du ing
neona al de elopmen (Supplemen a y Fig. S3). The lack o
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Lack o R-Ras and Vascula Pe meabili y in Re inopa hy IOVS jSep embe 2016 jVol. 57 jNo. 11 j4902
F
IGURE
2.
R-Ras is s ongly, bu selec i ely exp essed in he endo helial cells and pe icy es o blood essels in e ina. Oxygen-induced e inopa hy
was induced by exposing WT pups o 75% oxygen a P7 o 5 days and e u ning hem o no mal oom ai a P12. A e 5 days in no moxia (a P17),
he R-Ras exp ession was de e mined om he e ascula ized e inas by IHC and immuno luo escence (IF) using R-Ras–speci ic p ima y an ibody.
(A)
Rep esen a i e images o R-Ras exp ession in he OIR model. Mo e han 30% o he p e e inal blood essels a e nega i e o R-Ras (
a ow
),
whe eas blood essels in e ina a e R-Ras posi i e (
a owhead
).
Righ
, nega i e con ol (no p ima y an ibody). The e is also ain R-Ras exp ession
om o he e inal cells, p esumably om neu al cells in he e ina. 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. (
B–E
) Rep esen a i e
con ocal images showing he colocaliza ion o R-Ras (
g een
) and CD31 (
ed
) in he e ina o ozen sec ions a e immuno luo escence s aining. (
C
)
R-Ras is exp essed in he endo helial cells in he e ina and (
D
) also in he p e e inal blood essels. (
E
) Some o he p e e inal blood essels a e
nega i e o R-Ras. (
F
) R-Ras is exp essed in he pe icy es (NG2,
ed
) in he e ina. Some pe icy es o p e e inal blood essels a e nega i e o R-Ras
in OIR (
a ow
), bu (
G
) R-Ras is also exp essed in he pe icy es o p e e inal neo essels.
Scaleba s
: 100 lm (
A
,
B
), 20 lm (
C
–
G
).
in luence on de elopmen o e inal ascula u e by R-Ras is in
line wi h he abo e- epo ed low exp ession o R-Ras a he
ea ly s ages o e inal ascula de elopmen (Supplemen a y
Fig. S1).
Induc ion and Selec i e Disappea ance o R-Ras
F om he Pa hologic Neo ascula u e in OIR
Nex , he R-Ras exp ession p o ile in he OIR model was
analyzed using WT mice. Using qPCR, a 2.5- old inc ease o
R as
mRNA om no mal P17 o P17 OIR model e inas was
de ec ed. Using Wes e n blo analysis, a 7.5- old inc ease in R-
Ras p o ein le els was seen in OIR P17 e inas compa ed o
heal hy P17 e inas. A P12, he exp ession o R-Ras was
educed in OIR. The amoun o R-Ras exp ession is in line wi h
eg ession o he ascula u e a P12 and essel eg ow h a P17
in he OIR model (Fig. 1). Using R-Ras immunohis ochemis y,
R-Ras was ound o be localized mainly o he blood essels in
OIR e inas (Fig. 2, Supplemen a y Fig. S4). A ain R-Ras
exp ession was also de ec ed ou side o he e inal blood
essels in mouse e ina (Fig. 2A). This immunohis ochemical
signal mos p obably ep esen s low exp essions om e inal
neu onal cells, which a e known o exp ess R-Ras.
35
Using R-
Ras and endo helial cell (CD31), as well as R-Ras and pe icy e
(NG2), double-s aining, R-Ras exp ession was shown o be
con ined o endo helial cells and pe icy es in e inal essels
(Fig. 2). I appea ed ha all o he e inal blood essels
exp essed R-Ras, whe eas only 68% o he p e e inal neo essels
had R-Ras exp ession a P17 in he OIR model (Fig. 2A,
Supplemen a y Fig. S4). This inding was con i med by double
immuno luo escen imaging o he e ina showing some
p e e inal neo essels de oid o R-Ras exp ession in OIR (Figs.
2E, 2F). By cos aining he e inal issue sec ions o R-Ras and
endo helial cells, as well as o R-Ras and pe icy es, he R-Ras
exp ession was con i med o be con ined o bo h endo helial
c ell s an d pe i cy e s (Fig s. 2B–G ).

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F
IGURE
3.
Nei he e ascula iza ion no pa hologic neo ascula iza ion is a ec ed by R-Ras de iciency in he OIR model. Wild- ype and R-Ras KO
mice pups we e exposed o hype oxia as p e iously desc ibed, and e inas we e ha es ed a P17. A eas o ascula obli e a ion and pa hologic
neo ascula iza ion (meaning neo ascula u s) we e quan i ied om Isolec in IB
4
–s ained whole moun s using Adobe Pho oshop CS3. (
A
)
Rep esen a i e e inas o WT and R-Ras KO mice a P17. The e ascula iza ion a e was de e mined by quan i ying he a ascula a eas (
yellow
) in
e inal la moun s. The amoun o pa hologic neo ascula iza ion ( u s,
ed
) was also de e mined. (
B
) Summa y o quan i a i e analysis o ascula
obli e a ion (
yellow
) and neo ascula iza ion (
ed
). The a ascula and pa hologic neo ascula iza ion a eas we e measu ed, and esul s a e
ep esen ed as an a ea ela i e o WT a ea.
E o ba s
ep esen
6
95% con idence in e als. (WT:
n
¼
63, KO:
n
¼
48 e inas.)
R-Ras Does No Change he Hypoxic
Re ascula iza ion Ra e o he Re ina
As all o he an iangiogenic molecules used o ea neo ascula
e inal diseases ha e been shown o inhibi sp ou ing
angiogenesis induced in he OIR model,
36–38
he ole o R-Ras
in hypoxia-d i en neo ascula iza ion in he e ina was ex-
plo 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 R-Ras KO and WT mice. A e exposu e
o 75% oxygen be ween P7 and P12, e inas we e e alua ed a
P12. Typical o his model,
39
la ge a eas o he cen al ascula
ne wo k we e obli e a ed, wi h only a ew majo essels
emaining cen ally in bo h WT and R-Ras KO mice
(Supplemen a y Fig. S5). Quan i a i e analysis o obli e a ed
a eas in e ina con i med ha he e inal ascula u e in R-Ras
KO and WT mice a e a ec ed in a simila ashion by hype oxic
exposu e (Supplemen a y Fig. S5). Taken oge he , hese da a
sugges ha dele ion o R-Ras has no obse able e ec on
ascula eg ession and emodeling in he e ina in esponse o
hype oxic condi ions.
On e u n o no moxia, he a ascula and hypoxic cen al
e ina s imula es apid eg ow h o essels.
24
The a e o e inal
e ascula iza ion in WT and R-Ras KO e inas was de e mined
by quan i ying a ascula e inal a ea 5 days a e he mice we e
e u ned o no moxia. The e was no signi ican di e ence in
he a ascula e inal a ea be ween he WT and R-Ras KO,
indica ing ha he a e o e inal e ascula iza ion is simila
be ween he WT and R-Ras KO mice a P17 (Fig. 3). 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 in o 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.
24
P e e inal (i.e., pa hologic) neo ascula iza ion eaches
i s maximum in WT mice 5 days a e e u ning o no moxia (a
P17).
24
No signi ican di e ences in he numbe o p e e inal
neo ascula u s and clus e s we e ound be ween R-Ras KO
mice and WT mice (Fig. 3). The esul s indica e ha nei he he
hypoxia-induced e ascula iza ion a e o he obli e a ed a eas
in e ina no he a e o p e e inal neo ascula iza ion is
a ec ed by R-Ras dele ion.
R-Ras De icien Re inal Blood Vessels Ha e
Inc eased Pe meabili y in OIR
I has been ecen ly shown ha loss o R-Ras wo sens blood
essel s uc u e and pe usion and he pa hologic plasma
leakage in umo angiogenesis.
15,21
In o de o assess he ole
o R-Ras in con olling he ascula pe meabili y o he e inal
ascula u e, he OIR model was used. The ascula leakage was
quan i ied by measu ing EB dye ex a asa ion om e inal
essels. The R-Ras KO e inas had app oxima ely 100% inc ease
in he e inal EB leakage compa ed wi h he WT e inas in OIR
(
P
¼
0.0223 a 1 hou and
P
¼
0.0006 a 24 hou s; Figs. 4A–C).
No di e ence was de ec ed in he amoun o EB leakage when
compa ing no mal WT and R-Ras KO e inas (Supplemen a y
Fig. S6). To con i m he enhanced e inal leakage in R-Ras KO
animals in OIR, we also measu ed he amoun o IgG hea y
chain molecule accumula ion in he e ina, as his an ibody
subclass is no mally compa men alized wi hin he ci cula-
ion.
40
We could no de ec a di e ence in IgG leakage in he
no mal e inas be ween WT and R-Ras KO animals. A 4- old
inc ease in he amoun o IgG was de ec ed in he R-Ras KO
e inas o e he WT e inas in OIR as a sign o inc eased
ascula pe meabili y (
P
¼
0.0007; Fig. 4D). To ule ou he
possibili y ha he inc eased le els o IgG in he R-Ras KO OIR
e inas we e due o plasma cells, immunohis ologic s aining o
CD138/Syndecan-1 was pe o med. The e was no inc ease in
he numbe o plasma cells in KO e inas compa ed o WT in
he OIR samples (Supplemen a y Fig. S7).
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F
IGURE
4.
R-Ras de iciency inc eases essel pe meabili y in mouse OIR
model. Wild- ype and R-Ras KO mice pups we e exposed o hype oxia
as desc ibed p e iously. The EB dye was injec ed IP, and e inas and
blood samples we e ha es ed a e 1 hou and 24 hou s a P17. E ans
Blue concen a ions we e measu ed spec opho ome ically a 620 nm,
and EB concen a ion o each sample was calcula ed om a s anda d
cu e. Resul s a e exp essed as ela i e o EB concen a ion in he
plasma. (
A
) Rep esen a i e R-Ras KO and WT OIR e inas a e sys emic
injec ion o EB dye. In many o he R-Ras KO e inas, EB dye was isible,
whe eas mos o he WT e inas we e colo less. (
B
) S a is ical analysis
o a ep esen a i e expe imen shows a signi ican inc ease in ascula
leakage in R-Ras KO OIR e inas compa ed o WT OIR e inas al eady a
a 1-hou ime poin (
P
¼
0.0223,
*,
nonpa ame ic Mann-Whi ney
U

es , WT
n
¼
6, R-Ras KO
n
¼
8). (
C
) The e is a highly signi ican
di e ence in ascula leakage be ween R-Ras KO and WT mice a he
24-hou ime poin (
P
¼
0.0006,
***,
nonpa ame ic Mann-Whi ney
U

es ; WT
n
¼
20, R-Ras KO
n
¼
18).
E o ba s
ep esen SEM. To assess
he IgG p o ein accumula ion, OIR e inas we e ha es ed a P17,
p o eins we e ex ac ed and samples we e elec opho esed on g adien
gels, and Wes e n blo ing was done using an i-mouse IgG an ibody. (
D
)
Rep esen a i e images o immunoblo ing om whe e mouse IgG
hea y chain densi ies we e quan i ied and no malized agains GAPDH.
Rela i e compa ison o IgG hea y chain p o ein om WT OIR and R-
Ras KO OIR samples gi es a 3.5- old di e ence (
P
¼
0,0007,
***,
nonpa ame ic Mann-Whi ney
U
es ). The e is no di e ence in he IgG
p o ein le el be ween heal hy WT and KO mice a P17 (
P
¼
0.7879).
E o ba s
ep esen
6
95% con idence in e als. (WT:
n
¼
6, KO:
n
¼
6, WT OIR:
n
¼
14, KO OIR:
n
¼
16.)
R-Ras De iciency Se e ely Impai s Pe icy e
Co e age o Angiogenic Blood Vessels and Reduces
VE-Cadhe in Exp ession in OIR Model
Close in e ac ion be ween sp ou ing endo helium and pe i-
cy es is c ucial o essel ma u a ion and s abili y.
41,42
Pa hologic, angiogenic ascula u e in umo s is cha ac e ized
by insu icien pe icy e associa ion a ound he blood essels.
43
Consis en wi h his no ion, a signi ican educ ion in he
di ec physical con ac be ween pe icy es and he endo helium
o blood essels was ound in R-Ras KO e inas in he OIR
model a P17 (Figs. 5A–B). When he pe icy e co e age a ound
he ips o he sp ou ing angiogenic blood essels was
quan i ied, 40% educ ion in he pe icy e co e age was
eco ded in he R-Ras KO e inas o e he WT e inas (
P
¼
0.033; Fig. 5C).
Vascula endo helial–cadhe in is a membe o cadhe in
supe amily exp essed exclusi ely on endo helial cells.
21
I is a
c ucial ac o o adhe ens junc ions be ween he endo helial
cells.
15
R-Ras is known o s abilize adhe ens junc ions by
inhibi ing VEGF-induced VE-cadhe in in e naliza ion.
15
We
s udied he exp ession o VE-cadhe in in OIR model, and we
obse ed ha bo h he in ensi y o he VE-cadhe in immuno-
s aining and colocaliza ion o VE-cadhe in wi h CD31 we e
signi ican ly educed in KO mice compa ed o WT mice (Fig.
5D). Colocaliza ion analysis showed signi ican educ ion in
VE-cadhe in and CD31 colocaliza ion in R-Ras KO mice
compa ed o WT mice (o e lap coe icien acco ding o
Mande s:

¼
0.51 o WT, and
R
¼
0.43 o KO (
P
¼
0.04)).
Ou inding on VE-cadhe in exp ession is in line wha has been
epo ed in umo ascula u e o R-Ras KO and WT mice.
21
Reduced R–Ras Exp ession in he Neo essels o
Human Diabe ic Re inopa hy Co ela es Wi h
Inc eased Vascula Leakage and he Imma u e S a e
o he Neo essels
R-Ras exp ession was es ic ed exclusi ely o blood essels in
no mal human e ina, and i was exp essed in all blood essels
in no mal human e inas (Supplemen a y Fig. S8). To add ess
he ele ance o ou indings on he ole o R-Ras in he OIR
model o human ischemic e inopa hies, he pa hologic e inal
neo ascula memb anes ha de elop in human diabe ic
e inopa hy pa ien s we e s udied. These neo ascula mem-
b anes we e collec ed om pa ien s su e ing om ype I
diabe es, who had al eady de eloped ac ional e inal de ach-
men due o ib osis o neo ascula memb anes. Thus, he
samples ep esen he end s age o he disease, whe e
subs an ial amoun o ib osis is associa ed wi h neo essels,
bu hey s ill con ain egions wi h ac i e pa hologic angiogen-
esis. In he diabe ic neo ascula memb anes, R-Ras exp ession
was exclusi ely es ic ed o blood essels (Fig. 6). Like
pa hologic p e e inal neo essels in he OIR model, human
diabe ic neo ascula memb anes had a ying exp ession o R-
Ras (Fig. 6). Only app oxima ely 80% o he blood essels in he
human neo ascula memb anes exp essed any R-Ras p o ein a
all (Fig. 6C). To explo e whe he R-Ras exp ession has any
ela ion o he blood essel ma u i y, he human issue samples
we e double-s ained o R-Ras and VEGFR2, a ma ke o blood
essel imma u i y.
44,45
A s ong in e se co ela ion be ween
exp ession o he wo p o eins was iden i ied (Spea man’s q
¼
-
0.821,
P
¼
0.023,
R
2
¼
0.563); he highe he pe cen age o
imma u e blood essels (VEGFR2
þ
), he less R-Ras exp ession
he e was in he blood essels (Fig. 6).
To add ess whe he lack o R-Ras in imma u e blood essels
in luences ascula pe meabili y in human diabe ic neo ascu-
la memb anes, samples we e s ained o HSA. Samples wi h a
low pe cen age o R-Ras–exp essing blood essels showed a
s ong accumula ion o HSA ou side he essels, whe eas
samples wi h a high pe cen age o R-Ras–exp essing blood
essels had HSA s aining es ic ed mainly inside he essels
(Figs. 6A–B). The e was a s ong nega i e co ela ion be ween
a ea o ex a ascula HSA and he numbe o R-Ras–posi i e
essels (Spea man’s q:

¼ -
0.886,
P
¼
0.019,
R
2
¼
0.835); he
mo e ascula leakage ou side o he blood essels, he less R-
Ras exp ession was in he blood essels. Fu he mo e, we
could demons a e ha leaked, ex a ascula HSA was de ec ed
mainly a ound indi idual blood essels ha lack R-Ras (Fig. 7).
This was especially e iden in samples ha had R-Ras in he
majo i y o blood essels as ex a ascula HSA mainly
accumula ed a ound hose ew blood essels de oid o R-Ras
exp ession (Fig. 7).
DISCUSSION
The p esen s udy demons a es ha he small GTPase R-Ras
egula es he pa hologic pe meabili y o blood essels in
hypoxia-d i en angiogenesis wi hou al e ing he a e o
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F
IGURE
5.
Pe icy e co e age and VE-cadhe in exp ession is educed in R-Ras KO in he OIR model in angiogenic e inal blood essels. Oxygen-
induced e inopa hy was induced in WT and R-Ras KO mice as desc ibed p e iously. Re inas we e ha es ed a P17, and whole-moun e inas we e
double-s ained wi h Alexa Fluo –conjuga ed Isolec in IB
4
and wi h an an ibody agains he pe icy e ma ke NG2 p o eoglycan. Th ee-dimensional
images we e aken om he mos supe icial ascula plexus a he ips o he blood essels om he egion whe e essels g ow owa d he op ic
ne e. (
A
) Rep esen a i e images o WT and R-Ras KO blood essel endo helial cells (
ed
) su ounded by pe icy es (
g een
). (
B
) The majo i y o
images om WT mice e ina had a lo o pe icy es a ound he blood essels, whe eas he majo i y o pic u es aken om he KO e inal blood
essels we e lacking o had e y ew pe icy es. (
C
) Di ec con ac be ween endo helial cells and pe icy es was quan i ied by colocaliza ion analysis.
The esul is shown as a pe cen age o endo helial cell a ea colocalized wi h pe icy es in WT and R-Ras KO e inal blood essel ips. The pe icy e
co e age is signi ican ly educed in R-Ras KO animals compa ed o WT by 40% (
P
¼
0.033, *).
E o ba s
ep esen
6
95% con idence in e als.
(WT:
n
¼
15; R-Ras KO:
n
¼
15. Two pic u es we e aken om each e ina, and an a e age was calcula ed). The exp ession o VE-cadhe in was
s udied in he OIR model by s aining ozen c oss-sec ions o e ina wi h an ibodies agains VE-cadhe in and endo helial cells (CD31). The in ensi y
as well as he colocaliza ion o VE-cadhe in s aining wi h endo helial cells was quan i ied using BioImageXD. (
D
) Rep esen a i e images o VE-
cadhe in (
g een
) and CD31 (
ed
) in P17 OIR model in WT and R-Ras KO a e p esen ed. Colocaliza ion analysis showed signi ican educ ion in VE-
cadhe in and CD31 colocaliza ion in R-Ras KO mice compa ed o WT mice (o e lap coe icien acco ding o Mande s:

¼
0.51 o WT and
R
¼
0.43
o KO [
P
¼
0.04]),
n
¼
5 mice o WT and
n
¼
5 mice o KO.
e ascula iza ion o he hypoxic e ina. Fu he mo e, by
analyzing a se o p e e inal ascula memb anes ob ained
om diabe ic e inopa hy pa ien s, we we e able o demon-
s a e ha neo ascula memb anes wi h educed R-Ras
exp ession in imma u e essels display inc eased pa hologic
ascula leakage. Gi en ha inc eased ascula leakage is he
pa hognomonic ea u e in human diabe ic e inopa hy, ou
s udy implica es ha loss o R-Ras may ha e a pa hologic ole in
his disease.
R-Ras educed he pa hologic ascula pe meabili y in
ischemia-induced e inopa hy wi hou inhibi ing e inal angio-
genesis, whe he i was de elopmen al o induced by hypoxia
in OIR. This is a clea di e ence o all cu en a ailable
an iangiogenic he apies ha ha e been shown o unc ion in
OIR by inhibi ing sp ou ing angiogenesis.
36
Fu he mo e, he
mechanism o esis ance o he cu en ly a ailable an iangio-
genic he apies bo h in umo s and e inopa hy is ac ually
ela ed o he e adica ion o he neo essels, which, in u n,
wo sens he unde lying ischemia and d i es he o ma ion o
new, leaky blood essels by al e na i e molecula mecha-
nism.
13
Thus, he p oposed molecula mechanism o u u e
an iangiogenic he apies is one in which he angiogenic blood
essels a e
‘‘no malized’’
o s able ones o alle ia e he hypoxia
and s op he de imen al abe an ascula leakage.
46,47
Taken
wi h ea lie demons a ions o a causal ela ionship be ween R-
Ras exp ession and essel ma u a ion,
15,20,48,49
ou esul s
sugges po en ial u ili y o R-Ras in ea ing e inopa hies
in ol ing angiogenesis. Modula ing R-Ras exp ession ep e-
Downloaded F om: h p://io s.a ojou nals.o g/pd access.ashx?u l=/da a/Jou nals/IOVS/935707/ on 11/30/2016
Lack o R-Ras and Vascula Pe meabili y in Re inopa hy IOVS jSep embe 2016 jVol. 57 jNo. 11 j4906
F
IGURE
6.
Reduced R-Ras exp ession co ela es wi h leakage o human se um albumin in human diabe ic e inopa hy ascula u e, and i is
nega i ely co ela ed wi h VEGFR2 exp ession. P e e inal neo ascula memb anes ob ained om i ec omies om diabe ic e inopa hy pa ien s
we e analyzed by immunohis ochemis y o hei p o ein exp ession. Immunohis ochemical s aining om adjacen sec ions was done wi h an i-
CD31
þ
an i–R-Ras, an i-HSA, and an i–R-Ras
þ
an i-VEGFR2 an ibodies. The numbe o R-Ras–posi i e essels was calcula ed om R-Ras and CD31
double-s ained sec ions (
n
¼
7; 5–9 di e en sec ions analyzed om each sample). Co ela ion analysis be ween R-Ras and VEGFR2 was done om R-
Ras
þ
VEGFR2 double-s ained sec ions and compa ed o CD31 s aining om adjacen sec ions (
n
¼
7; 5–9 di e en sec ions analyzed om each
sample). Vascula memb anes wi h s ong R-Ras exp ession show limi ed ex a ascula s aining o HSA (
A
), whe eas samples wi h weak R-Ras
exp ession show s ong s aining o HSA ou side he blood essels (
B
).
Scaleba s
: 100 lm. The e is a s ong in e se co ela ion be ween HSA-
posi i e ex a ascula a ea and he pe cen age o R-Ras–posi i e neo essels (Spea man’s q:

¼-
0.886,
P
¼
0.019, *,
R
2
¼
0.835,
n
¼
6 pa ien s) (
D
).
(
C
) App oxima ely 20% o he neo essels do no exp ess any R-Ras a all (R-Ras–posi i e essels 81
6
10%, mean
þ
SD,
n
¼
6 pa ien s). Da a a e
shown as a box plo wi h median and 95% con idence in e al. (
E
,
F
) Double-s aining o R-Ras and VEGFR2 shows an in e se co ela ion
(Spea man’s q:

¼-
0.821,
P
¼
0.023,
R
2
¼
0.563) be ween he numbe o R-Ras–exp essing and VEGFR2-posi i e blood essels. When a majo i y o
blood essels has R-Ras exp ession, a majo i y o blood essels a e nega i e o VEGFR2 exp ession, whe eas he VEGFR2 exp ession is opposi e
when e y ew blood essels exp ess R-Ras.
Scaleba s
: 200
lm.
sen s a no el he apeu ic app oach ha may be capable o
add essing pa hologic ascula pe meabili y wi hou simul a-
neously wo sening he unde lying pa hobiology o he
disease— hypoxia.
The di e ences be ween he mechanisms o R-Ras and
o he an iangiogenic he apies a e e idenced by a ecen
epo implica ing ac i a ed Ras signaling as a key media o o
pa hologic neo ascula iza ion induced by many cy okines and
g ow h ac o s, including VEGF, in blinding neo ascula eye
diseases.
22
Wes enskow e al.
22
epo ed ha abe an Ras
signaling can be inhibi ed by a molecule called p120RasGAP.
The inhibi o y unc ion o p120RasGAP on Ras-d i en e inal
neo ascula iza ion was shown o ake place h ough he
inhibi ion o sp ou ing angiogenesis.
22
In con as , R-Ras
unc ions in he e ina p ima ily o s abilize ascula u e and
p e en leakage and does no inhibi sp ou ing angiogenesis in
OIR. The mechanis ic di e ences be ween R-Ras and p120Ras-