ACE2 and Ang-(1-7) Confer Protection Against Development of Diabetic Retinopathy

Transcription

1 originl rticle ACE nd Ang-(-7) Confer Protection Aginst Development of Dietic Retinopthy Amrish Verm, Zhiying Shn, Bo Lei, Lihui Yun, Xun Liu, Tkhiko Nkgw, Mri B Grnt 5, Alfred S Lewin, Willim W Huswirth, Mohn K Rizd nd Qiuhong Li Deprtment of Ophthlmology, University of Florid, Ginesville, Florid, USA; Deprtment of Physiology nd Functionl Genomics, University of Florid, Ginesville, Florid, USA; The First Affilited Hospitl of Chongqing Medicl University, Chongqing Key Lortory of Ophthlmology, Chongqing Eye Institute, Chongqing, Chin; Division of Renl Disese nd Hypertension, University of Colordo Denver, Auror, Colordo, USA; 5 Deprtment of Phrmcology nd Therpeutics, University of Florid, Ginesville, Florid, USA; Deprtment of Moleculr Genetics nd Microiology, University of Florid, Ginesville, Florid, USA Despite evidence tht hyperctivity of the vsodeleterious xis (ACE/ngiotensin II (Ang II)/AT receptor) of the renin ngiotensin system (RAS) is ssocited with the pthogenesis of dietic retinopthy (DR) use of the inhiitors of this xis hs met with limited success in the control of this pthophysiology. We investigted the hypothesis tht enhncing the locl ctivity of the recently estlished protective xis of the RAS, ACE/ Ang-(-7), using deno-ssocited virus (AAV)-medited gene delivery of ACE or Ang-(-7) would confer protection ginst dietes-induced retinopthy. Genes expressing ACE nd Ang-(-7) were cloned in AAV vector. The effects of oculr AAV-ACE/Ang-(-7) gene trnsfer on DR in dietic enos / mice nd Sprgue Dwley (SD) rts were exmined. Dietes ws ssocited with pproximtely tenfold nd greter thn threefold increses in the rtios of ACE/ACE nd ATR/ Ms mrna levels in the retin respectively. Introculr dministrtion of AAV-ACE/Ang-(-7) resulted in significnt reduction in dietes-induced retinl vsculr lekge, cellulr cpillries, infiltrting inflmmtory cells nd oxidtive dmge in oth dietic mice nd rts. Our results demonstrte tht DR is ssocited with impired lnce of retinl RAS. Incresed expression of ACE/Ang-(-7) overcomes this imlnce nd confers protection ginst DR. Thus, strtegies enhncing the protective ACE/Ang-(-7) xis of RAS in the eye could serve s novel therpeutic trget for DR. Received Octoer ; ccepted June ; pulished online July. doi:./mt..55 INTRODUCTION Dietic retinopthy (DR) is the most common dietic vsculr compliction, nd despite recent dvnces in therpeutics nd mngement, DR remins the leding cuse of severe vision loss in people under the ge of. The renin ngiotensin system (RAS) plys vitl role in the crdiovsculr system. Angiotensin II (Ang II), peptide hormone of the RAS, hs een known to regulte vriety of hemodynmic physiologicl responses, including fluid homeostsis, renl function, nd contrction of vsculr smooth muscle. Furthermore, Ang II is known to medite multitude of other effects, such s the induction of rective oxygen species, cytokines, growth fctors, nd collgen synthesis. 5 In ddition to circulting RAS, incresing evidence implictes the involvement of the locl RAS in retinl vsculr dysfunctions. Vrious components of RAS hve een detected in the eye. Elevted levels of renin, prorenin, nd Ang II hve een found in ptients with DR. In fct ACE inhiitors (ACEi) nd ngiotensin receptor lockers (ARBs) hve een shown to improve dietes-induced retinl vsculr, neuronl, nd glil dysfunction. Recent clinicl studies hve lso clerly demonstrted eneficil effects of RAS inhiition in oth type nd type dietic ptients with retinopthy. 7 Despite these positive outcomes, RAS lockers re not completely retinoprotective nd retinopthy still progress to lte stge. This could e ttriuted to the existence of locl Ang II formtion nd tht the RAS lockers re unle to cross the lood-retin rrier (BRB) in concentrtion sufficient to influence the locl RAS in the eye. In ddition, incresing evidence suggests tht Ang II cn e generted vi multiple pthwys tht my not e locked y clssic ACEi. 5 Furthermore, dditionl components of RAS tht contriute to end-orgn dmge, such s the receptor for renin nd prorenin, hve een recentlyidentified. Activtion of prorenin/pro/renin receptor signling pthwy cn initite RAS cscde independent of Ang II. Discovery of ngiotensin-converting enzyme (ACE) hs resulted in the estlishment of novel xis of the RAS involving ACE/Ang-(-7)/Ms. 7, This vsoprotective xis countercts the trditionl prolifertive, firotic, proinflmmtory nd hypertrophic effects of the ACE/Ang II/ATR xis of the RAS. 7 The importnce of the vsodeleterious xis of the RAS [ACE/Ang II/ATR] in crdiovsculr disese, s well s in dietes nd dietic complictions, is well estlished since ACEi nd ARBs re leding therpeutic strtegies. 9 However, the impct of the vsoprotective xis of the RAS remins poorly understood. 7, 5 The concept tht shifting the lnce of the RAS towrds the vsodiltory xis y ctivtion of ACE or its product, Ang-(-7) is eneficil hs een supported y mny studies in crdic, pulmonry, nd vsculr firosis. 7, Indeed, ACE/Ang-(-7) ctivtion is now considered to e criticl prt of the eneficil ctions of ACEi Correspondence: Qiuhong Li, Deprtment of Ophthlmology, University of Florid, Ginesville, Florid -, USA. E-mil: qli@ufl.edu vol. no., jn.

2 ACE/Ang-(-7) Protects Retin From Dietes-induced Vsculr Dmge nd ARB drugs. 7, We hypothesized tht n imlnce in the vsoprotective versus vsodeleterious xis of the RAS, prticulrly within the oculr tissue, would result in the development nd progression of DR nd tht enhncing the protective xis of ACE/ Ang-(-7) t the tissue level would directly counterct the effects of Ang II, regrdless of its intrcellulr sources of formtion. In this study, we tested this hypothesis y exmining the retinl RAS gene expression during the progression of dietic retinopthy nd evluting the effects of incresed expression of ACE/ Ang-(-7) in the retin using deno-ssocited virus (AAV)- medited gene trnsfer. We showed tht intrvitrel dministrtion of AAV vector expressing ACE or Ang-7 peptide reduced dietes-induced retinl pthophysiology in two rodent models. RESULTS Expression of the retinl RAS genes in the enos / mouse retins during the progression of dietes We hve previously shown tht dietes induced y streptozotocin (STZ) tretment in enos / mice results in more severe, ccelerted retinopthy thn dietes in enos +/+ nimls. Thus, our first ojective ws to compre retinl mrna levels of the RAS genes in control nd dietic nimls during the progression of dietes. We oserved three to ten fold increses in the mrna levels of the vsodeleterious xis of the RAS (ngiotensinogen, renin, pro/ renin receptor, ACE nd AT receptor sutypes) following STZ tretment (Figure ). In contrst, there ws ~% reduction in ACE mrna following n initil rief stimultory response. As result ACE/ACE mrna rtio ws incresed y tenfold, while ATR/Ms rtio ws incresed y threefold following month of dietes (Figure ). The Ms mrna level ws slightly incresed erly in dietes (Figure ), followed y more thn 5% decrese y month fter dietes. The reson for this discrepncy etween mrna levels nd protein ctivity of ACE is still uncler. It could e due to chnges in post-trnscriptionl modifictions, or protein degrdtion/stility. And further studies will e needed to elucidte the underlying mechnisms. Nevertheless, these oservtions were our first indiction tht DR is ssocited with shifting in lnce of the retinl RAS towrd vsodeleterious xis. Chrcteriztion of AAV vectors expressing ACE nd Ang-(-7) AAV vector expressing the secreted form of humn ACE ws constructed under the control of the chick- -ctin (CBA) promoter (Figure ). This secreted form of ACE hs een previously chrcterized nd shown to e enzymticlly ctive. Since Ang-(-7) peptide contins only 7 mino cids nd smll peptides re usully difficult to express in mmmlin cells, we hve designed n expression construct in which the Ang-(-7) peptide is expressed s prt of the secreted fusion green fluorescent protein (GFP) protein, which is susequently cleved upon secretion. The expression of the fusion sgfp-fc-ang-(-7) is under the control of the CBA promoter in the AAV vector (Figure ) nd ws confirmed y trnfecting the HEK9 cells using this plsmid DNA (Figure ). To ensure tht the fusion protein ws indeed secreted, proteins isolted from the culture superntnts s well s cell lystes from trnsfected, shm-trnsfected or untrnsfected cells were nlyzed y western lotting (Figure ). ACE ATR Renin Angiotensinogen ACE/ACE ACE Mss spectrometry nlysis of Ang (-7) peptide in superntnt smples of HEK9 cells trnsfected with the sgfp-fc- Ang-(-7) plsmid DNA ws lso performed. The Ang-(-7) peptide ws detectle in superntnt isolted from cells trnsfected with sgfp-fc-ang-(-7) plsmid DNA, ut ws not detectle in smples isolted from un-trnsfected cells, or cells trnsfected with the control plsmid expressing the cytoplsmic GFP (dt not shown). Intrvitrel dministrtion of AAV-Ang-(-7) resulted in roust trnsduction of retinl cells primrily inner retinl lyer (Figure c f). This ws ssocited with n increse in oth cellulr nd secreted Ang-(-7) (Figure g h). Similrly, ACE protein level ws incresed in the retin following trnsduction with AAV-ACE (Figure g). Oculr gene delivery of ACE/Ang-(-7) vi the AAV vector in the retin results incresed ACE ctivities nd Ang-(-7) peptide levels Induction of dietes resulted in more thn fivefold increse in ACE ctivities in the retins of enos / mice, wheres ACE ATR/Ms ATR Renin receptor Ms receptor ATR/Ms NDM D DM M DM Figure Rel-time reverse trnscriptse (RT)-PCR nlysis of retinl mrna for renin-ngiotensin system genes. Vlues on y-xis represent fold difference compred to ge-mtched nondietic retinl smples for ech gene t ech time point ( dy nd month fter induced dietes). DM, dietic; NDM, nondietic. At lest four eyes were nlyzed t ech time point. P <. (versus NDM group). Moleculr Therpy vol. no. jn. 9

3 ACE/Ang-(-7) Protects Retin From Dietes-induced Vsculr Dmge CBA promoter hace SVpoly(A) c d Signl peptide Ang-(-7) CBA promoter GFP SVpoly(A) FC CBA promoter GFP SVpoly(A) ~ kd ~7 kd e f g kd 75 kd Fusion GFP Cleved GFP No injection ACE-AAV ~5 kd 7 kd 5 kd h (Ang -7) (pg/ g retinl protein) Uninjected Ang-7-AAV injected Figure Construction nd chrcteriztion of deno-ssocited virus (AAV) vectors expressing ngiotensin-converting enzyme (ACE) nd Ang-(-7). () Mps of the AAV vector expressing the humn ACE gene (hace) nd the AAV vector expressing Ang-(-7) gene. The Ang-(-7) peptide is expressed s prt of fusion protein, nd cleved upon secretion t the furin clevge (FC) site. CBA, CMV-chicken- -ctin promoter;, inverted terminl repet. A control vector contins coding region for the secreted green fluorescent protein (GFP) without Ang-(-7) peptide coding sequence. () Expression nd clevge of the fusion protein. In cultured HEK9 cells trnsfected with the plsmid sgfp-fc-ang-(-7), or infected with AAV-sGFP-FC-Ang-(-7), there ws roust expression of GFP s expected. Proteins isolted from cell lystes contined single protein nd with moleculr weight ~ kd, s predicted for the precursor (fusion protein), ut culture superntnts contined two protein nds ( kd nd 7 kd), indicting tht the secreted protein is cleved t the furin clevge site s predicted. (c f) Trnsduction of mouse retin with AAV vector expressing sgfp-fc-ang-(-7) nd hace. A single intrvitrel injection of μl AAV vector ( 9 vg/eye) resulted in efficient trnsduction of inner retinl cells, primrily retinl gnglion cells. (c) Low mgnifiction of cross section of mouse eye tht received AAV-sGFP-FC-Ang-(-7) injection. (d) Higher mgnifiction of the sme eye. (e) A retinl whole mount showing GFP expression. (f) Higher mgnifiction of the sme retinl whole mount. Br = μm in d nd f. (g) Western lot of proteins isolted from n uninjected eye nd n eye injected with AAV-ACE (top) nd AAV- sgfp-fc-ang-(-7) (ottom) compred to moleculr weight stndrd (right lne). (h) Ang-(-7) peptide levels in the retin with nd without AAV-sGFP-FC-Ang-(-7) injection. There ws more thn tenfold increse in Ang-(-7) peptide level detected y using n Ang-(-7) specific EIA kit (Bchem, Sn Crlos, CA) in retins receiving injection of AAV-sGFP-FC Ang-(-7). INL, inner nucler lyer; IPL, inner plexiform lyer; OPL, outer plexiform lyer; PR, photoreceptor; RGC, retinl gnglion cells. ctivity ws reltively unchnged (Figure ). AAV-ACE injected retins showed more thn twofold increse in ACE enzymtic ctivities (Figure ) nd this ws ssocited with reduced level of Ang II nd incresed Ang-(-7) peptide level (Figure ), ut hd mrginl effect on ACE ctivity (Figure ). Injection of AAV-Ang-(-7) hd no effect on ACE ctivity, ut significntly decresed ACE ctivity (Figure ). We lso determined Ang II nd Ang-(-7) peptide levels. STZ induced dietes resulted in more thn twofold increse in Ang II levels wherese Ang-(-7) level ws un-chnged in the retins of enos / mice (Figure ). The increse of Ang II ws completely normlized in retins injected with AAV-ACE ut ws unchnged in retins injected with AAV-Ang-(-7) vector (Figure ). Locl oculr tretment with either AAV-ACE or AAV- Ang-(-7) vector hd no effects on ody weight, lood glucose nd lood pressure in dietic enos / mice (Supplementry Tle S). Incresed ACE/Ang-7 expression in the retin reduced dietes-induced retinl pthophysiology We investigted if elevted retinl expression of ACE or Ang-(-7) would overcome vsodeleterious effect of ACE/ATR xis nd prevent the development of dietes-induced retinopthy. The effect of incresed ACE nd Ang-(-7) expression on retinl vsculr permeility ws evluted y fluorescein isothiocynte-leled lumin extrvstions nd quntified y mesuring fluorescent intensity in seril sections from nondietic, untreted, ACE vol. no. jn.

4 ACE/Ang-(-7) Protects Retin From Dietes-induced Vsculr Dmge M DM enos DM enos + ACE DM enos + Ang-(-7) ACE ACE ACE/ACE Ang II levels Ang-(-7) levels 5 NDM DM DM + DM + ACE NDM DM DM + Ang-(-7) Ang-(-7) DM + ACE Figure Angiotensin-converting enzyme (ACE), ACE ctivities, nd ngiotensin peptide levels in the retins. () ACE nd ACE enzymtic ctivities nd ACE/ACE rtios in nondietic (NDM), month dietic (M DM), nd month dietic enos / retins treted with AAV-ACE/ Ang-(-7). Vlues re expressed s fold differences compred with ge-mtched nondietic group. P <. (versus untreted DM group, N = / group). () Ang II nd Ang-(-7) peptide levels in nondietic (NDM), month dietic (M DM), nd month dietic enos / retins treted with AAV-ACE/Ang-(-7), mesured y ELISA using commercil kit. P <. (versus untreted DM group). Vlues represent fold difference compred with ge-mtched nondietic group. Three retins were pooled for ech mesurement, ech mesurement ws done in duplictes, nd three seprte pools were verged for ech group. Reltive fluorescein intensity nd Ang -7 treted dietic enos / mice. Induction of dietes for month in enos / mice resulted in twofold increse in vsculr permeility, this pthophysiology ws significntly reduced in dietic retins tht received ACE/Ang-(-7) vector tretments (Figure ), ut not in the retins tht received tretment with control vector which contined the coding sequence for secreted GFP without Ang-(-7) or ACE (dt not shown). Dietes induced incresed infiltrting CD5 positive mcrophges nd ctivtion of CD positive microglil cells; this Untreted DM enos ACE-DM enos Ang-7-DM enos Figure Effects of oculr tretments with ngiotensin-converting enzyme (ACE) nd Ang-(-7)-AAV on retinl vsculr permeility in dietic enos / mice. Retinl vsculr permeility ws evluted y fluorescein isothiocynte (FITC)-leled lumin extrvstions nd quntified y mesuring the fluorescent intensity in seril sections from enos / mice t month fter induced dietes. Dt re presented s men ± SD from six eyes in ech group. P <. (versus untreted DM group). DM, dietes; NDM, nondietes. ws significntly reduced in eyes treted with ACE nd Ang-(-7) expression vectors (Figure 5). Induction of dietes for months in enos / mice resulted in greter thn tenfold increse in the formtion of cellulr cpillries nd this ws significntly reduced in dietic retins which received ACE/Ang-(-7) vector tretments (Figure ). Furthermore, this increse in the level of ACE lso prevented the sement memrne thickening in dietic enos / retin (Supplementry Figure S). Finlly, we used STZ-induced dietes in SD rts s n dditionl niml model of dietes to provide further conceptul vlidtion. We oserved more thn fivefold increse in the numer of cellulr cpillries in STZ-induced dietic rt retins t month of dietes. This increse ws lmost completely prevented y gene delivery of either ACE or Ang-(-7) (Figure 7). Agin oculr tretment with either AAV-ACE or AAV- Ang-(-7) vector hd no effects on ody weight, lood glucose nd lood pressure in dietic SD rts (Supplementry Tle S). Incresed expression of ACE/Ang-(-7) reduced oxidtive dmge in dietic retin Dietes nd its complictions re ssocited with incresed oxidtive stress. We ssessed oxidtive dmge mesuring the levels of thiorituric cid-rective sustnces ( mrker for oxidtive dmge) in the retin. Dietes induced significnt increse in thiorituric cid-rective sustnces (Figure ) in Moleculr Therpy vol. no. jn.

5 ACE/Ang-(-7) Protects Retin From Dietes-induced Vsculr Dmge / M DM enos / c / M DM enos / ACE-M DM enos / Ang-7-M DM enos / ACE-M DM enos / Ang-7-M DM enos / Numer of CD5 cells/section DM enos - untreted DM enos - ACE DM enos - Ang -7 d Numer of CD cells/section DM enos - untreted DM enos - ACE DM enos - Ang -7 Figure 5 Intrvitrel dministrtion of ACE or Ang-(-7)-AAV reduces dietes-induced oculr inflmmtion. () Representtive imges of CD5 + cells in the nondietic, untreted, ACE, nd Ang-(-7)-treted dietic enos / mouse retins t month fter induced dietes. () Quntifiction of CD5 + inflmmtory cells in the retins from the nondietic, untreted, ACE, nd Ang-(-7)-treted dietic enos / mouse retins t month fter induced dietes. (c) Representtive imges of CD + cells in the nondietic, untreted, ACE, nd Ang-(-7)-treted dietic enos mouse retins t month fter induced dietes. (d) Quntifiction of CD + inflmmtory cells in the retins from the nondietic, untreted, ACE, nd Ang-(-7)-treted dietic enos mouse retins t month fter induced dietes. N = for ech group. P <. (versus untreted DM group). Br = 5 μm. INL, inner nucler lyer; IPL, inner plexiform lyer; ONL, outer nucler lyer; OPL, outer plexiform lyer; RGC, retinl gnglion cells. enos / mouse retins (Figure ). This increse ws completely prevented y AAV-ACE or Ang-(-7) tretment. Similr results were lso otined in SD rt retins (Figure ). DISCUSSION We demonstrte tht ll the genes within the RAS re expressed in the retin, consistent with vrious previous reports (reviewed in ref. nd references therein), nd tht the expression levels of genes in the vsoconstrictive rm of RAS (renin, ACE, ATR) re highly elevted in dietic retins. In contrst, there is n initil increse in the expression of genes in the vsoprotective xis (ACE nd MAS) erly in dietes; this is reduced during the dietes progression, thus tipping the lnce towrd more vsoconstrictive, proinflmmtory, hypertrophic effects of RAS medited y ACE/Ang II/ATR xis. This imlnce of locl RAS is ssocited with incresed ACE ctivity nd Ang II levels in the dietic retins nd unchnged ACE ctivity nd Ang-(-7), lthough the ACE nd Ms receptor mrna levels re reduced. Furthermore, we demonstrte tht enhnced expression of either ACE or Ang-(-7) vi AAV vector medited gene delivery in the retin prevents dietes-induced retinl vsculr permeility, thickening of sement memrne, retinl inflmmtion, formtion of cellulr cpillries, nd oxidtive dmge in oth mouse nd rt models of dietic retinopthy. More importntly, these eneficil effects occur in the sence of systemic improvement of glucose, lood pressure, oth of which re elevted in enos / mice (Supplementry Tle S), nd other dietic complictions, suggesting tht dysregultion of locl RAS plys significnt role in the pthogenesis of dietic retinopthy, nd cn e modulted loclly to restore the lnce etween the two counter-cting rms y enhncing the ACE/Ang-(-7)/MAS xis. These oservtions provide conceptul support tht enhncing ACE/ Ang-(-7) xis mye n effective strtegy for the tretment of DR. Although vrious components of RAS hve een detected in retin, our study is the first to exmine the expression levels vol. no. jn.

6 ACE/Ang-(-7) Protects Retin From Dietes-induced Vsculr Dmge / ACE-M DM enos / M DM enos / NDM SD rt Ang-(-7)-M DM enos / ACE-M SD rt DM enos-untreted DM enos-ace DM enos-ang-(-7) Figure Evlution of cellulr cpillry formtion in untreted nd AAV-ACE/Ang-(-7) treted retins of dietic mice. Tretments with ACE nd Ang -7 vectors in the dietic enos / mouse retins reduced cellulr cpillries. () Representtive imges of trypsindigested retinl vsculr preprtions from nondietic enos /, untreted, ACE, nd Ang-(-7)-treted dietic enos / mouse retins ( month fter induced dietes). Arrows indicte the cellulr cpillries. () Quntittive mesurements of cellulr cpillries. The vlues on y-xis represent the numer of cellulr cpillries/mm retin. DM, dietes; NDM, nondietes. N =. P <. (versus untreted DM group). of ll known RAS genes during the progression of dietes in the enos / mice, which exhiit ccelerted retinopthy. We show tht incresed expression of genes in the vsoconstrictive, proinflmmtory xis of RAS (ACE, ATR, renin, renin receptor) occur erly, dys fter STZ-induced dietes. We hve previously shown tht incresed retinl vsculr permeility nd gliosis re lredy detectle t this time point in dietic enos / mouse retin, suggesting tht locl hyperctivity of the deleterious xis (ACE/Ang II/ATR) my contriute to these pthologicl chnges. We lso mesured ACE nd ACE ctivities in dietic enos / mouse retin. In contrst to previous report which showed tht ACE enzyme ctivity ws decresed, nd ACE enzyme ctivity ws incresed in dietic rt retins, we found tht ACE ctivity is highly incresed in these dietic retins, wheres ACE ctivity remined unchnged. This discrepncy my e due to the difference in niml models or the time points exmined. The importnce of the vsodeleterious xis of the RAS (ACE/ Ang II/ ATR) in crdiovsculr disese, s well s in dietes nd Moleculr Therpy vol. no. jn. Ang-(-7)-M DM SD rt Numer of cellulr cpillries/mm Numer of cellulr cpillries/mm M DM SD rt DM rt-untreted DM rt-ace DM rt-ang-7 Figure 7 Evlution of cellulr cpillry formtion in untreted nd ACE/Ang-(-7) AAV vectors treted retins of dietic SD rts. () Representtive imges of trypsin-digested retinl vsculr preprtions from nondietic SD rt, untreted, ACE, nd Ang-(-7)treted dietic SD rt retins ( month fter induced dietes). () Quntittive mesurements of cellulr cpillries. The vlues on y-xis represent the numer of cellulr cpillries/mm retin. DM, dietes; NDM, nondietes. N =. P <.(versus untreted DM group). dietic complictions, is well estlished since ACEi nd ARBs re leding therpeutic strtegies.9 However, the impct of the vsoprotective xis of the RAS remins poorly understood, prticulrly in the eye. The concept tht shifting the lnce of the RAS towrds the vsodiltory xis y ctivtion of ACE or its product, Ang-(-7) is eneficil hs een supported y mny studies in crdic, pulmonry, nd vsculr firosis.7, 5 In greement with these studies, we now show tht incresed expression of either ACE or Ang-(-7) is protective in oth dietic enos / mouse nd rt model of dietic retinopthy. The protective effect of ACE ppers to e result of reduced Ang II, y ctlyzing its conversion to Ang-(-7), thus incresing the level of Ang-(-7), or comintion of oth. Indeed, in the AAV-ACE injected retin, the dietes-induced elevtion of Ang II ws reduced nd this ws ssocited with incresed level of Ang-(-7). However, the fct tht incresed Ang-(-7) expressed from AAV vector in the retin ws eqully protective despite high Ang II levels suggests tht Ang-(-7) lone cn directly counterct the effect of high retinl Ang II levels in dietes. This is consistent with well-estlished effects of Ang-(-7).5

7 ACE/Ang-(-7) Protects Retin From Dietes-induced Vsculr Dmge TBARs ( mol/l/mg totl protein) 7 5 TBARs ( mol/l/mg totl protein) NDM DM DM+ACE DM+Ang-(-7) NDM DM DM+ACE DM+Ang-(-7) Figure Thiorituric cid-rective sustnces (TBARs) levels in enos / mouse retins (left) nd SD rt retins (right). Dietes resulted in incresed TBARs levels in oth enos / mouse retins ( month dietes) nd SD rt retins (t month dietes). These increses were prevented y AAV-ACE/Ang-(-7) tretments. DM, dietes; NDM, nondietes. N = /group. P <.(versus untreted DM). It is interesting to note tht ACE overexpression ws ssocited with reduced Ang II nd incresed Ang-(-7) levels s expected, ut hd no effect on ACE ctivity. In contrst, overexpression of Ang-(-7) hd no effect on endogenous ACE ctivity, ut significntly reduced ACE ctivity. Prdoxiclly, despite reduced ACE ctivity in AAV-Ang-(-7) injected retin, Ang II level remined high. It is possile tht other enzymes/pthwys esides ACE mye involved in the formtion of Ang II. One such cndidte is the chymse, which hs een detected in vsculr system nd other tissues including eye. Another cndidte pthwy is the receptor for prorenin nd renin (pro/renin). Binding of pro/ renin to its receptor, pro/renin receptor, cuses its prosegment to unfold, therey ctivting prorenin so tht it is le to generte ngiotensin peptides tht stimulte the Ang II-dependent pthwy. Considering the fct tht retin contins high level of prorenin, which is further incresed in ptients with dietic retinopthy, this pthwy my contriute to the incresed Ang II level tht is oserved in dietes. The existence of multiple pthwys for Ang II formtion t the tissue level my explin the limited eneficil effects of clssic RAS lockers, nd lso lend the support for the notion tht enhncing the protective xis of RAS (ACE/ Ang-(-7)/Ms) my represent more effective strtegy for tretment of dietic retinopthy nd other dietic complictions. AAV vector medited gene therpy for oculr diseses hs een studied in niml models for more thn decde. Reports focusing on retinl therpy include wide vriety of retinl degenertive niml models of corresponding humn retinopthies, s well s the therpeutic effects of AAV-vector medited expression of neuroprotective, nti-poptotic, nti-ngiogenic gents in the retin. 7 In view of recent clinicl trils, gene therpy hs emerged s vile pproch nd my ecome tretment options for rnge of diseses in the future. In prticulr, when considering tht dietic individul experiences oculr complictions for decdes, therpeutic strtegy tht is long-lsting nd tht does not require repeted nd frequent dministrtion is desirle. Thus, the delivery of ACE nd/or Ang-(-7) could serve s novel gene therpy trget for DR in comintion with existing strtegies to control hyperglycemi nd hypertension. In summry, our oservtions demonstrte tht dietesinduced retinopthy is ssocited with n imlnce in the locl RAS s result of ctivtion of the deleterious xis (ACE/Ang II/ ATR) nd decrese in the vsoprotective xis (ACE/Ang--7/ Ms) of the RAS. Restoring this lnce y ACE or Ang-(-7) overexpression provides profound protective effects ginst this pthophysiology. Thus, these results provide proof-of-principle tht enhncing ACE/Ang-(-7)/MAS xis my represent promising pproch for treting DR nd other dietic complictions. MATERIALS AND METHODS Animls nd Experimentl procedures. All the niml procedures dhere to protocols pproved y the University of Florid Institutionl Animl Cre nd Use Committee. Two niml models of DR were studied: the STZ-induced dietic enos / mouse model, which hs een recently shown to develop ccelerted retinopthy, nd the STZ-induced dietic SD rt model. Breeding pirs of enos / mice were purchsed from Jckson Lortories (Jckson Lortories, Br Hror, ME) nd mle SD rts ( weeks old) were purchsed from Hrln Lortories (Indinpolis, IN). All nimls were mintined t the Animl Cre Service t the University of Florid. All procedures dhere to ARVO sttement for the Use of Animls in Ophthlmic nd Vision Reserch, nd pproved protocols y the Animl Cre nd Use Committee of the University of Florid. Animls were fed stndrd lortory chow nd llowed free ccess to wter in n ir-conditioned room with -hour light drk cycle. Dietes ws induced y single intrperitonel injection of freshly prepred STZ solution (5 mg/kg ody weight in mmol/l citrte uffer, ph.5) for rts. Two consecutive intrperitonel injections of STZ ( mg/kg ody weight, freshly mde in.5 mol/l citrte uffer, ph.5) were used to induce dietes in mice (mle, ~ weeks old). Dietes ws confirmed one week fter STZ tretment y mesuring the lood glucose level (defined > mg/dl) using the FreeStyle glucomonitor nd test strip ccording to the mnufcturer s instruction. Mesurements of mrna levels of retinl renin-ngiotensin genes nd inflmmtory cytokines. Totl RNA ws isolted from freshly dissected retins using Trizol Regent (Invitrogen, Crlsd, CA) ccording to mnufcturer s instruction. Reverse trnscription ws performed using Enhnced Avin HS RT-PCR kit (Sigm, St Louis, MO) following mnufcturer s instructions. Rel time PCR ws crried out on rel time therml cycler (icycler, Bio-Rd Life Sciences) using iqtm Syer Green Supermix (Bio-Rd Life Sciences, Hercules, CA). The threshold cycle numer (Ct) for rel-time PCR ws set y the cycler softwre. Optiml primer concentrtion for PCR ws determined seprtely for ech primer pir. Ech rection ws run in duplicte or in triplicte, nd rection tues with trget primers nd those with GAPDH/Actin primers were lwys included in the sme PCR run. To test the primer efficiencies, the one-step reverse trnscriptse-pcr ws run with ech trget primer. Reltive quntifiction ws chieved y the comprtive ΔΔCt method. The reltive increse/decrese (fold-induction/repression) of mrna of trget X in the experimentl group (EG) ws clculted using the control group s the vol. no. jn.

8 ACE/Ang-(-7) Protects Retin From Dietes-induced Vsculr Dmge clirtor: ΔΔCt, where ΔΔ Ct is {Ct.x [EG] Ct. GAPDH [EG]} {Ct.x [control] Ct. GAPDH [control]}. Primer sequences used in this study re shown in Supplementry Tle S. AAV vectors nd other procedures. An AAV vector contining secreted form of humn ACE gene under the control of the chimeric CMV/chicken -ctin promoter ws constructed. We previously showed tht the secreted ACE protein expressed from lentivirl vector is enzymticlly ctive. The construction nd chrcteriztion of Ang-(-7) peptide expression vector is descried in Supplementry Dt. To determine the protective role of ACE nd Ang-(-7), the vectors were dministered y intrvitrel injection two weeks efore STZ induction of dietes. Only one of eyes for ech niml ws injected nd the contrlterl eye ws used s control. Generl prmeters (ody weight, glucose levels, nd lood pressure) of these nimls with nd without tretments re shown in Supplementry Tle S for mice nd Supplementry Tle S for rts in Supplementry Mterils nd Methods. Retinl vsculr permeility ws evluted t month fter induction of dietes, the loss of retinl cpillries ws evluted t months fter induction of dietes for enos / mice, nd month fter induction of dietes for SD rts. These time points were chosen sed on the previously chrcteriztion of retinl vsculr chnges. At lest six nimls were used for ech type of end-point ssys. Methods for retinl vsculr evlution (permeility, cellulr cpillries, immunohisto-chemistry nlysis) re essentilly the sme s descried previously, nd re presented in Supplementry Dt. Sttisticl nlysis. All vlues re presented s men + SD. Pired Student s t-test ws used to ssess significnce etween two groups. One-wy nlysis of vrince followed y the post hoc Tukey (Fisher s protected lest significnt difference) test ws used to ssess sttisticl significnce etween multiple groups. Differences were considered significnt t P <.5. SUPPLEMENTARY MATERIAL Figure S. Trnsmission electron microgrphs of retinl cpillries of dietic retin with nd without vector tretment. Tle S. Primers used for rel-time RT-PCR nlysis. Tle S. Generl prmeters of enos / mice. Tle S. Generl prmeters of SD rts. Mterils nd Methods. Supplementry Dt. ACKNOWLEDGMENTS This work ws supported y grnt from Americn Dietes Assocition, NIH grnts EY, HL nd HL59, Foundtion Fighting Blindness, Juvenile Dietes Reserch Foundtion, nd Reserch to Prevent Blindness, Inc. W.W.H. is the co-founder of AGTC Inc. A ptent ppliction for Ang-(-7) expression vector y University of Florid is pending. The other uthors declred no conflict of interest. REFERENCES. Cheung, N, Mitchell, P nd Wong, TY (). Dietic retinopthy. Lncet 7:.. 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