Angiotensin(1-7) and ACE2, “The Hot Spots” of Renin-Angiotensin System, Detected in the Human Aqueous Humor

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. REFERENCES [1] Fyh quis F, Saijonmaa O. Renin-angio ensin sys em e isi ed. J In e n Med 2008; 264: 224-36. [2] Paul M, Poyan Meh A, K eu z R. Physiology o local enin angio ensin sys ems. Re Physiol Re 2006; 86: 747-803. [3] K amkowski K, Mogielnicki A, Buczko W. The physiological signi icance o he al e na i e pa hways o angio ensin II p oduc ion. J Physiol Pha macol 2006; 57: 529-39. [4] Danse AHJ, De kx FHM, Admi aal PJJ e al. Angio ensin le els in he eye. In es Oph halmol Vis Sci 1994; 35: 1008-18. [5] Sa askan E, Lo le KU, Meie F, e al. 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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. [15] Wang RF, Podos SM, Mi ag TW, Yokoyoma T. E ec o CS-088, an angio ensin AT1 ecep o an agonis , on in aocula p essu e in glaucoma ous monkey eyes. 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J Ocul Pha macol The 2014; 30: 170-80. [22] Osusky R, Nussbe ge J, Ams u z C, Flamme J, B unne HR. Indi idual measu emen s o angio ensin II concen a ions in aqueous humo o he eye. Eu J Oph halmol 1994; 4: 228-33. [23] Fe ão FM, La a LS, Lowe J. Renin-angio ensin sys em in he kidney: Wha is new? Wo ld J Neph ol 2014; 3: 64-76. [24] Shio a N, Saegusa Y, Nishimu a K, Miyazaki M. Angio ensin II- gene a ing sys em in dog and monkey ocula issues. Clin Exp Pha macol Physiol 1997; 24: 243-8. [25] Vaajanen A, Kalesnykas G, Vapaa alo H, Uusi alo H. The exp ession o Mas- ecep o o he enin-angio ensin sys em in he human eye. G ae es A ch Clin Exp Oph halmol 2015 [Epub ahead o p in ]. 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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