Send O de s o Rep in s o ep in s@ben hamscience.ae
28 The Open Oph halmology Jou nal, 2015, 9, 28-32
1874-3641/15 2015 Ben ham Open
Open Access
Angio ensin(1-7) and ACE2, “The Ho Spo s” o Renin-Angio ensin
Sys em, De ec ed in he Human Aqueous Humo
Me i Holappa1, Ja kko Valjakka1 and Anu Vaajanen*,2,3
¹BioMediTech, Uni e si y o Tampe e, Tampe e, Finland
²Depa men o Oph halmology, Tampe e Uni e si y Hospi al, Tampe e, Finland
³SILK, Depa men o Oph halmology, School o Medicine, Uni e si y o Tampe e, Tampe e, Finland
Abs ac : Backg ound: The main pu pose o he s udy was o es ablish whe he essen ial componen s o he enin-
angio ensin sys em (RAS) exis in he human aqueous humo .
Me hods: Fo y- i e pa ien s ≥ 60 (74±7) yea s o age unde going ca a ac su ge y a Tampe e Uni e si y Hospi al we e
andomly selec ed o he p ospec i e s udy. The exclusion c i e ion was he use o o al an ihype ensi e medicine ac ing
ia enin-angio ensin sys em. Aqueous humo samples we e aken a he beginning o no mal ca a ac ex ac ion. The
samples we e ozen and s o ed a -80 °C. The concen a ions o in aocula endogenous RAS componen s Ang(1-7),
ACE2, and ACE1 we e measu ed using ELISA.
Resul s: Concen a ion medians o Ang(1-7), ACE2, and ACE1 in he aqueous humo we e: Ang(1-7) 4.08 ng/ml, ACE2
2.32 ng/ml and ACE1 0.35 ng/ml. The concen a ions we e signi ican ly highe in glaucoma ous han in non-
glaucoma ous eyes, ACE1 (p=0.014) and Ang(1-7) (p=0.026) s non-glaucoma ous eyes.
Conclusions: Ang(1-7), ACE2 and ACE1 a e ound in he human aqueous humo . The obse a ions a e consis en wi h
he concep ion ha local issue-RAS exis s in he human eye and i migh ha e a ole in he con ol o in aocula p essu e.
Keywo ds: Angio ensin (1-7), angio ensin con e ing enzyme 2, angio ensin II, angio ensin con e ing enzyme 1, aqueous
humo , glaucoma, enin-angio ensin sys em.
INTRODUCTION
The sys emic enin-angio ensin sys em (RAS) con ols
luid olume, elec oly e balance and blood p essu e (BP)
homeos asis [1]. RAS is also ega ded as a issue-speci ic
egula o y sys em accoun ing o local e ec s and long- e m
changes in di e en o gans [2, 3]. Many pep ides and
enzymes o RAS ha e al eady been de ec ed in he human
eye [2, 4, 5] and hey a e e en sugges ed o ha e a ole in he
pa hogenesis o di e en ocula diseases including glaucoma
[6]. One o he majo known isk ac o s o glaucoma is
inc eased in aocula p essu e (IOP) [7-9] which is a ne sum
o homeos a ic balance be ween aqueous humo o ma ion
and ou low.
I has ecen ly been epo ed ha o ally adminis e ed
an ihype ensi e d ugs can also educe IOP [10]; o
example, o al angio ensin con e ing enzyme (ACE)
inhibi o (cap op il) [11] and he AT1- ecep o blocke
(ARB) [12] (losa an) ha e been shown o lowe IOP in bo h
non-glaucoma ous and glaucoma ous pa ien s. In animal
s udies, ACE inhibi o s [13, 14] ARBs [15, 16], enin
inhibi o s [17] and angio ensin (1-7) [18] ha e been epo ed
o lowe IOP, e en when hey a e locally adminis e ed.
These indings imply ha a local in aocula RAS may be
*Add ess co espondence o his au ho a he Depa men o
Oph halmology, Tampe e Uni e si y Hospi al, P.O. Box 2000, 33521
Tampe e, Finland; Tel: +358-3-31164852; E-mail: anu. aajanen@ imne . i
in ol ed in he egula ion o IOP [19, 20]. Recen ly, o he
b oade heo ies ha e been published on RAS in ol emen
in he pa hogenesis o glaucoma [6, 21]. So a , only AngII
and ACE1 ha e been iden i ied in he human aqueous humo
[6, 22]. RAS is known o consis o o e wen y pep idases,
close o wen y angio ensin pep ides, and a leas six
ecep o s [23]. AngII-ACE1-angio ensin 1 ecep o ype
(AT1R)-axis oge he wi h Ang(1-7)-ACE2-Mas- ecep o
(MasR)-axis a e seen as he main pa hways o RAS ha may
media e he apeu ic bene i s. The main pu pose o his s udy
was o de ec he le els o Ang(1-7), ACE2 and ACE1
quali a i ely and quan i a i ely in he human aqueous humo .
MATERIALS AND METHODOLOGY
The s udy was unde aken in he Depa men o
Oph halmology in he Uni e si y Hospi al o Tampe e,
Finland be ween 28 h Feb ua y and 8 h Ap il 2014. The s udy
con o ms o he Wo ld Medical Associa ion Decla a ion o
Helsinki and he E hical P inciples o Medical Resea ch
In ol ing Human Subjec s, and ecei ed app o al om he
Regional E hics Commi ee a Tampe e (ETL R14010).
In o med consen was ob ained om all pa icipan s. Pa ien s
o e 60 yea s o age unde going ca a ac su ge y we e
included. The pa ien s we e selec ed andomly om he
ca a ac ope a ion lis and hey we e ope a ed upon by he
same su geon. The only exclusion c i e ion was he use o
o al an ihype ensi e medicine ac ing ia enin-angio ensin
sys em. Toge he , 45 pa ien s we e ope a ed upon; 15 o
Ang(1-7) and ACE2 in he Human Eye The Open Oph halmology Jou nal, 2015, Volume 9 29
hem had been diagnosed wi h glaucoma acco ding o
Finnish E idence- Based Guidelines [9]. All he
glaucoma ous pa ien s we e ocula no mo ensi e because o
con inuous an iglaucoma medica ion o p e ious glaucoma
su ge y. Thus all he eyes independen ly o medica ion we e
no mo ensi e, and he di ision o glaucoma ous/non-
glaucoma ous was based on he his o y o he pa ien s.
Medical eco ds (age, gende , medica ions and IOP) we e
egis e ed du ing he p eope a i e isi . IOP was measu ed
using a ebound onome e (Ica e®, Ica e Finland Oy, Van aa,
Finland). Blood p essu e was measu ed h ee consecu i e
imes p io o su ge y in si ing posi ion and he a e age o
he measu emen s was calcula ed.
An addi ional ele en pa ien s we e ope a ed upon; six o
hem we e aking o al ACE inhibi o medica ion o high
blood p essu e and i e o hem we e on ARBmedica ion.
These pa ien s we e no included in he main s udy esul s,
bu hei an e io chambe RAS componen concen a ions
a e shown in Fig. (1).
Aqueous humo samples (0.05-0.35 ml) we e aken in he
beginning o ou ine ca a ac su ge y. The liquid was
collec ed in o Eppendo ubes and immedia ely chilled in an
icebox. A e he ope a ion, he collec ed samples we e
ozen and s o ed a -80°C wi hin 2 h. No p o ease/pep idase
inhibi o s we e added in he collec ion and s o age o he
samples. The samples we e analyzed using comme cially
a ailable enzyme-linked immunoso ben assays (ELISA)
due o hei applicabili y o simul aneously assess he a ge
molecule concen a ions in a la ge numbe o samples.
Ang(1-7) was measu ed using he Human Angio ensin(1-7)
Elisa ki (MyBioSou ce, San Diego, CA, USA) wi h a
de ec ion limi o 0.1 ng/ml. ACE2 was measu ed using he
Human ACE2 ELISA ki (Bos e Immunoleade , Pleasan on,
CA, USA) wi h a de ec ion limi o <10 pg/ml, ACE1 using
he Human ACE ELISA ki (Bos e Immunoleade ) wi h a
de ec ion limi o <5 pg/ml. AngII was also measu ed using
he Angio ensin II ELISA ki (Enzo Li e Sciences,
Fa mingdale, NY, USA) wi h a de ec ion limi o 4.6 pg/ml.
The ki s we e designed o usage wi h human se um, plasma,
cell cul u e supe na es, body luid o issue homogena es.
The assay p ocedu es and assays we e conduc ed acco ding
o manu ac u e s’ ins uc ions. Only Angio ensin II ELISA
ki has been shown o ha e c oss- eac i i y, bu i is o no
ele ance. In o he ELISA ki s c oss- eac ions we e
negligible. All assays we e done blinded. Resul s a e shown
in ng/ml.
The da a om he ELISA assays we e analyzed using
Mic oso Excel and SPSS S a is ics o Windows e . 21.0.
(IBM Co p, A monk, NY, USA). Nonpa ame ic Mann-
Whi ney es and Spea man's Co ela ion we e used o da a
wi h skewed dis ibu ion and he esul s a e shown as median
wi h uppe and lowe qua iles, whe eas independen sample
- es and Pea son’s Co ela ion we e used o no mally
dis ibu ed da a. The esul s a e shown as mean ±SD. The
le el o signi icance was se a ˂0.05 ( wo- ailed) in all
s a is ical es s.
RESULTS
Fo y-six aqueous humo samples we e analyzed om 45
subjec s wi h an a e age age o 74±7 (mean ± SD) yea s. O
hese, 27 (60 %) we e emale and 18 (40 %) male. Fi een
we e diagnosed wi h glaucoma and 30 we e non-
glaucoma ous. O e all demog aphics o he glaucoma ous
and non-glaucoma ous subg oups a e p esen ed in Table 1.
Table 1. Demog aphics and in acame al concen a ions o
Ang(1-7), ACE2 and ACE1. Fo no mally
dis ibu ed da a esul s a e shown as mean ±SD, o
skewly dis ibu ed da a median wi h uppe and
lowe qua iles. The small olume o some samples
did no allow all measu emen s.
Non-Glaucoma ous
(n = 30) †
Glaucoma ous
(n = 15)
p-Value
Age
74 ± 7
75 ± 8
0.843
Gende (M/W)
10/20
8/7
IOP
15 ± 4
16 ± 6
0.658
Sys olic BP
163 ± 21
166 ± 26
0.692
Dias olic BP
88 ± 10
92 ± 11
0.335
Ang(1-7)
3.81
4.53
0.026*
(3.69-4.59)
(4.03-6.21)
(n = 31)
(n = 15)
ACE2
2.26
2.96
0.409
(1.19-6.02)
(1.91-12.33)
(n = 20)
(n = 14)
ACE1
0.27
0.48
0.014*
(0.23-0.39)
(0.33-0.79)
(n = 20)
(n = 12)
†Numbe o pa ien s: n=30, numbe o eyes: n=31. * p- alue<0.05.
Ang(1-7), angio ensin (1-7); ACE1, -2, angio ensin- con e ing enzyme 1, -2; BP,
blood p essu e; IOP, in aocula p essu e; M, men; W, women.
Median aqueous humo (n=46) Ang(1-7), ACE2 and
ACE1 concen a ions we e: 4.08 ng/ml (Q1-Q3; 4.00-4.92),
2.32 ng/ml (Q1-Q3; 2.58-7.53) and 0.35 ng/ml (Q1-Q3;
0.30-0.51), espec i ely. None o he samples showed
measu able le els o AngII. The Ang(1-7) and ACE1
concen a ions we e signi ican ly highe in glaucoma ous
han in non-glaucoma ous eyes (p=0.026 and p=0.014). See
Table 1. Indi idual aqueous humo concen a ions o Ang(1-
7), ACE2 and ACE1 in non-glaucoma ous and glaucoma ous
pa ien s a e p esen ed in Fig. (1).
Age, IOP- and BP- alues did no di e be ween he wo
subg oups (Table 1). Wi h one excep ion, no signi ican
di e ences be ween men and women we e ound in Ang(1-
7), ACE2 o ACE1 concen a ions in he subg oups (Table
2). The signi ican co ela ions we e in he non-
glaucoma ous g oup ACE1 s age and ACE1 s ACE2 bo h
gende s oge he . In glaucoma ous pa ien s no co ela ions
we e ound.
Glaucoma pa ien s (n=15) used di e en opically
adminis e ed an iglaucoma d ugs. Pa ien s who used
p os aglandin analogues had highe Ang(1-7) and ACE1
concen a ions s pa ien s wi h no medica ion (p=0.012 and
p=0.028 espec i ely). Also, he use o a combina ion o be a
blocke and ca bonic anhyd ase inhibi o was associa ed
wi h highe ACE1 concen a ion (p=0.035). No s a is ically
30 The Open Oph halmology Jou nal, 2015, Volume 9 Holappa e al.
signi ican di e ence was de ec ed be ween pa ien s using
o he glaucoma medica ions.
DISCUSSION
The p esen s udy was aimed o de ec he cen al
componen s o RAS in he human aqueous humo . In
addi ion possible di e ences be ween glaucoma ous and
non-glaucoma ous eyes we e s udied. We showed ha
endogenous Ang(1-7) and ACE1 and ACE2 a e p esen in
he aqueous humo o he human eye. The glaucoma ous
eyes ha e highe le els o ACE1 s non-glaucoma ous eyes.
This would sugges a ole o ACE1 in IOP balance in he
de elopmen o glaucoma. Fu he mo e, ACE1 le els we e
associa ed wi h highe ACE2 concen a ions in non-
glaucoma ous eyes, his o se ing each o he 's e ec s on
IOP. In e es ingly, in he glaucoma ous subjec s age did no
co ela e wi h he aqueous humo concen a ions o any o
he RAS componen s, while in non-glaucoma ous eyes
inc easing age was wi h highe ACE1 concen a ions.
Aqueous humo Ang(1-7), ACE1 and ACE2 le els did no
di e be ween he gende s in ei he o he subg oups. BP and
IOP alues we e no associa ed wi h highe RAS componen
concen a ions. High BP alues measu ed jus be o e su ge y
we e likely o esul om he anxie y and ea ha pa ien s
usually eel p io o an ope a ion. On he o he hand, all
glaucoma ous pa ien s we e unde ea men and he e o e
had no mal IOP alues.
The lack o measu able le els o AngII in aqueous humo
samples may be explained by he absence o
p o ease/pep idase inhibi o s in he collec ion and s o age o
he samples. Thus AngI (DRVYIHPFHL) and AngII
(DRVYIHPF) pep ides can be clea ed o sho e angio ensin
pep ides [24]; o AngIII (RVYIHPF) o Ang(1-9)
(DRVYIHPFH) o Ang(1-7) (DRVYIHP) [2]. Fo example,
p olyl endopep idase and p olyl ca boxypep idase can
hyd olyze AngII o Ang(1-7). These al e na i e pa hways o
angio ensin deg ada ion sys em a e possible. In p e ious
s udies [4] in aocula AngII de ec ion measu emen s we e
pe o med in pools consis ing o di e en samples. I is also
possible ha he sensi i i y o he used AngII assay was oo
weak in his s udy. Un o una ely, he e is no e e ence o
li e a u e showing a igo ous assessmen o hese ki s.
Usually, ELISA me hods a e quali a i e and quan i a i e bu
hey need highly speci ic and sensi i e an ibodies. Because
he sensi i i y o he assays is s ic ly limi ed by he a ini y
be ween an ibodies, pep ides, and p o eins, i is no possible
in p ac ice o accu a ely alida e o une assays.
In e es ingly, subjec s who used p os aglandin analogues
as glaucoma medica ion had highe Ang(1-7) and ACE1
concen a ions. In addi ion, he use o a combina ion o be a
Fig. (1). Indi idual aqueous humo concen a ions o Ang(1-7), ACE2 and ACE1 in non-glaucoma ous (whi e open ci cles) and
glaucoma ous (black spo s) pa ien s. Compa ison wi h Mann-Whi ney es , *p<0.05. Pa ien s using ACE inhibi o s (plus ma ks) o AT-
ecep o blocke s (x ma ks) a e also shown; hese pa ien s we e excluded om he da a in de e mina ion o he median concen a ions
(ho izon al lines). Fo abb e ia ions see Table 1.
Ang(1-7) and ACE2 in he Human Eye The Open Oph halmology Jou nal, 2015, Volume 9 31
blocke + ca bonic anhyd ase inhibi o was associa ed wi h
highe ACE1 concen a ions. Due o he limi ed numbe o
pa ien s using glaucoma medica ions in he p esen s udy,
hese obse a ions equi e u he con i ma ion.
CONCLUSION
Angio ensin(1-7) and ACE2, he “ho spo s” in he enin-
angio ensin sys em, a e ound in he human aqueous humo .
This suppo s he assump ion ha in aocula RAS may be
in ol ed in he egula ion o IOP. This heo y is u he
s ongly suppo ed by ou e y ecen obse a ion [25] on he
exp ession o Mas- ecep o s in he e ina and especially in
he an e io pa o he human eye.
CONFLICT OF INTEREST
The au ho s con i m ha his a icle con en has no
con lic o in e es .
ACKNOWLEDGEMENTS
The au ho s wish o hank he g ea eam in he ope a ing
hea e o he Eye Cen e a Tampe e Uni e si y Hospi al,
especially nu ses Ms Anja Ko piaho, Ms Michiko F anzen
and Ms Si pa A onen. The au ho s hank he Päi ikki and
Saka i Sohlbe g Founda ion, he Eye Founda ion, he
Glaucoma Resea ch Founda ion Lux and he Founda ion o
Clinical Chemis y Resea ch o suppo ing he s udy.
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Table 2. Co ela ions be ween RAS componen s (ng/ml), sex, age, IOP and BP. The small olume o some samples did no allow all
measu emen s. Fo abb e ia ions see Table 1.
Non- Glaucoma ous
Glaucoma ous
Ang(l-7)
n = 31
ACE2
n = 20
ACE1
n = 20
Ang(l-7)
n = 15
ACE2
n = 14
ACE1
n = 12
ACE2
p- alue
0.156
0.770
Cc†
0.330
0.100
ACE1
p- alue
0.361
0.025*
0.347
0.947
Cc†
0.216
0.595
-0.298
-0.023
Gende
Women
3.80
1.81
0.25
4.03
3.29
0.51
Men
4.05
2.88
0.40
4.82
3.22
0.41
p- alue
0.210
0.119
0.052
0.031*
0.101
0.497
Age
p- alue
0.128
0.960
0.027*
0.292
0.214
0.621
Cc†
0.259
-0.011
0.901
0.241
0.290
-0.152
lOP
p- alue
0.338
0.897
0.600
0.282
0.617
0.465
Cc†
-0.178
-0.029
-0.125
-0.297
0.170
0.234
Sys olic BP
p- alue
0.823
0.875
0.502
0.794
0.519
0.829
Cc†
-0.042
-0.036
0.160
0.077
0.218
-0.070
Dias olic BP
p- alue
0.769
0.452
0.332
0.567
0.729
0.444
Cc†
-0.055
0.169
0.229
-0.168
-0.118
0.244
†Cc. co ela ion coe icien .
*p- alue < 0.05.
32 The Open Oph halmology Jou nal, 2015, Volume 9 Holappa e al.
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Recei ed: Decembe 30, 2014 Re ised: Ma ch 2, 2015 Accep ed: Ma ch 2, 2015
© Holappa e al.; Licensee Ben ham Open.
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