Furin deficiency in myeloid cells leads to attenuated revascularization in a mouse-model of oxygen-induced retinopathy

Con en s lis s a ailable a ScienceDi ec Expe imen al Eye Resea ch jou nal homepage: www.else ie .com/loca e/yexe Fu in deficiency in myeloid cells leads o a enua ed e ascula iza ion in a mouse-model o oxygen-induced e inopa hy Ma ia Vähä upa a , Zuze Ma inez Co do a a,b , Ha lan Ba ke a , Saa a Ai omäki a,b , Hannu Uusi alo a,c,∗ , Te o A.H. Jä inen a,d , Ma ko Pesu a,b,e , Hannele Uusi alo-Jä inen a,c a Facul y o Medicine & Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland b Immuno egula ion, Ins i u e o Biosciences and Medical Technology (BioMediTech), Uni e si y o Tampe e, Tampe e, Finland c Eye Cen e, Tampe e Uni e si y Hospi al, Tampe e, Finland d Depa men s o Musculoskele al Diso de s, Tampe e Uni e si y Hospi al, Tampe e, Finland e Depa men s o De ma ology, Tampe e Uni e si y Hospi al, Tampe e, Finland ARTICLE INFO Keywo ds: Angiogenesis Fu in Mac ophage Oxygen-induced e inopa hy model Hypoxia ABSTRACT Ischemic e inopa hy is a ision- h ea ening disease associa ed wi h ch onic e inal inflamma ion and hypoxia leading o abno mal angiogenesis. Fu in, a membe o he p op o ein con e ase amily o p o eins, has been implica ed in he egula ion o angiogenesis due o i s essen ial ole in he ac i a ion o se e al angiogenic g ow h ac o s, including ascula endo helial g ow h ac o -C (VEGF-C), VEGF-D and ans o ming g ow h ac o - β(TGF- β). In he p esen s udy, we e alua ed exp ession o u in in he e ina and i s ole in e inal angiogenesis. As bo h inflamma ion and hypoxia con ibu e o angiogenesis, he ole o u in was e alua ed using myeloid-cell specific u in knockou (KO) mice (designa ed LysMC e- u (fl/fl) ) bo h in de elopmen al e - inal angiogenesis as well as in hypoxia-d i en angiogenesis using he oxygen-induced e inopa hy (OIR) model. In he e ina, u in exp ession was de ec ed in endo helial cells, mac ophages and, o some ex en , in neu ons. The a e o angiogenesis was no diffe en in LysMC e- u (fl/fl) mice when compa ed o hei wild- ype li e ma es du ing de elopmen . In he OIR model, he e ascula iza ion o e ina was significan ly delayed in LysMC e- u (fl/fl) mice compa ed o hei wild- ype li e ma es, while he e was no compensa o y inc ease in he p e e inal neo ascula iza ion in LysMC e- u (fl/fl) mice. These esul s demons a e ha u in exp ession in myeloid cells plays a significan ole in hypoxia-induced angiogenesis in e ina. 1. In oduc ion Ischemic e inopa hies, such as e inopa hy o p ema u i y (ROP), p oli e a i e diabe ic e inopa hy (PDR), and e inal ein occlusion (RVO) a e majo causes o isual impai men and blindness in in- dus ialized coun ies (Fong e al., 2004; Jonas e al., 2017; Reynolds, 2014). Re inal ischemia induces he s abiliza ion o a ansc ip ion ac o hypoxia inducible ac o -1α(HIF-1α), which u ns on he ex- p ession o a la ge numbe o angiogenic genes such as ascula en- do helial g ow h ac o (VEGF) (Campochia o, 2015). This leads o e inal neo ascula iza ion associa ed wi h i eous hemo hages and fib osis o neo ascula memb anes causing ac ional e inal de ach- men and, ul ima ely, blindness (Campochia o, 2015). Re inal ischemia is also associa ed wi h inflamma o y mani es a- ions. Hypoxia a ac s mac ophages in o hypoxic a eas (Mi amu a e al., 2005; Tsu sumi e al., 2003) whe e he hypoxia-ac i a ed mac- ophages and mic oglia, he immune cells o he e ina, elease no only p oinflamma o y bu also angiogenic cy okines. In diabe ic e inopa hy (DR), bo h in ading mac ophages and esiden mic oglia ha e been implica ed in he pa hogenesis o he e inopa hy e inopa hy (Abcouwe , 2012). O no e, mac ophages ha e been shown o be he main sou ce o VEGF-C in he e ina. In he e ina, VEGF-C signaling pa hway egula es he b anching o blood essels (Tammela e al., 2011). VEGF-C is ini ially syn hesized as an inac i e p ecu so and e- qui es ac i a ion by a p op o ein con e ase sub ilisin/kexin (PCSK) amily membe u in o become biologically ac i e (Sieg ied e al., 2003b). P op o ein con e ase sub ilisin/kexin (PCSK) amily enzymes a e a amily o nine p o eases (PCSK1-2, u in, PCSK4-7, MBTPS1, PCSK9) ha clea e and con e hei imma u e a ge p o eins in o biologically ac i e o ms by ca alyzing endop o eoly ic clea age a a a ge si e ypically made up o he basic amino acids a ginine and lysine (Tu peinen e al., 2013). Acco dingly, PCSK enzymes play a key eg- ula o y ole in a mul i ude o biological e en s go e ned by he g ow h h ps://doi.o g/10.1016/j.exe .2017.10.013 Recei ed 4 Janua y 2017; Recei ed in e ised o m 1 Sep embe 2017; Accep ed 11 Oc obe 2017 ∗ Co esponding au ho Facul y o Medicine & Li e Sciences, 33014, Uni e si y o Tampe e, Finland. E-mail add ess: llhauus@u a.fi(H. Uusi alo). Expe imen al Eye Resea ch 166 (2018) 160–167 A ailable online 13 Oc obe 2017 0014-4835/ © 2017 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/BY-NC-ND/4.0/). T ac o s (Tu peinen e al., 2013). Among he PCSK amily membe s, u in is a ubiqui ously exp essed p o o ypical se ine endop o ease e- cen ly implica ed o play a key ole in a ious biological p ocesses such au oimmuni y and inflamma ion, and a ious human diseases (Oksanen e al., 2014; Pesu e al., 2008; Tu peinen e al., 2013; Vähä upa e al., 2016a). In fib o ascula issue om PDR pa ien s, u in exp ession was shown o co-localize wi h one o i s clea age a ge s, a (p o)- enin e- cep o p o ein, whose exp ession le el is ele a ed in PDR and asso- cia ed wi h angiogenic ac i i y, sugges ing ha u in clea age has a ole in DR (Kanda e al., 2012). In addi ion o VEGF-C (Kha ib e al., 2010; Sieg ied e al., 2003a), u in is also equi ed o he ac i a ion o a la ge numbe o o he p oangiogenic g ow h ac o s, such as VEGF-D (McColl e al., 2007), pla ele de i ed g ow h ac o s A and B (PDGF-A and –B) (Sieg ied e al., 2003b, 2005), bone mo phogene ic p o ein-4 (BMP-4) (Cui e al., 1998) and ans o ming g ow h ac o -β(TGF-β)(Dubois e al., 2001). On he o he hand, he exp ession o u in i sel can be induced by hypoxia, as i is one o he a ge genes o HIF-1α(Ma e al., 2017; McMahon e al., 2005; Sil es i e al., 2008). Despi e being linked o angiogenesis, he unc ions o u in in hese p ocesses a e s ill poo ly known, bu he pheno ype o he o al u in knockou (KO) mice, le hali y du ing emb yonal de elopmen due o ca dio ascula de ec s, sugges ed blood essel specific unc ions (Roeb oek e al., 1998; Scamuffa e al., 2006). This no ion was confi med by endo helial spe- cific u in KO ha also led o emb yonal dea h due o ascula de ec s, and by he endo helial cells lacking u in ha we e unable o g ow ex i o (Kim e al., 2012). As endo helial cells lacking u in canno g ow a all (Kim e al., 2012), we decided o elucida e he ole o u in on e inal angiogenesis by add essing he exp ession o u in in he e ina and by assessing he effec o myeloid cell-de i ed u in on e inal angiogenesis using u in condi ional KO mice (myeloid cell-specific u in KO; LysMC e- u (fl/fl) ). The effec o myeloid cells-de i ed u in on angiogenesis was s udied in de elopmen al e inal angiogenesis, as well as in hypoxia-induced an- giogenesis in a mouse-model o oxygen-induced e inopa hy (OIR), which is he mos widely used model o ischemic e inopa hies (Conno e al., 2009; Smi h e al., 1994). In he OIR model, bo h e- c ui ed, blood-de i ed mac ophages as well as he esiden mac o- phages o he e ina, mic oglia, ha e been shown o pa icipa e in he egula ion o angiogenesis (Checchin e al., 2006; Gao e al., 2016; Ka aoka e al., 2011; Zhou e al., 2015). 2. Resul s 2.1. Fu in is exp essed in mac ophages and e inal mic oglia Using da a om published genome-wide exp ession s udies, he e inal-cell specific E-GEOD-33089 expe imen and he much wide sea ching FANTOM p ojec , we ex ac ed exp ession da a o u in in cells ela ed o e ina. In he la ge , bu a less specific, FANTOM p ojec cDNA analysis o u in exp ession showed he highes alues in monocy e de i ed mac ophages (Fig. 1A). In he e inal cell-specific expe imen , based on 155 samples analyzed by mic oa ay, he highes u in exp ession was ound in mic oglia (Fig. 1B). 2.2. Fu in is exp essed in he e ina du ing de elopmen and in he OIR model To u he s udy he ole o u in on e inal angiogenesis in i o,we fi s de e mined a empo al and spa ial exp ession p ofile o u in in WT mice du ing de elopmen and in he OIR model. Immunohis ochemis y showed a ew cells wi h weak exp ession o u in in e ina a P4 (pos na al day 4), and he ea e he exp ession o u in was sub- s an ially s onge om P7 onwa ds a all s udied ime poin s du ing de elopmen (Fig. 1C). In he OIR model, s ong exp ession o u in was de ec ed in he ascula laye s o he e ina a P14 and P17 (Fig. 1D). Using double-immunohis ochemis y o u in and o endo helial cells ma ke CD31 as well as o u in and mac ophage ma ke F4/80, u in exp ession was localized o endo helial cells (Fig. 1E–F) in he e ina and in he p e e inal ascula u s as well as o mac ophages su - ounding blood essels (Fig. 1G–H) in OIR e inas. In addi ion o hese cells, u in immuno eac i i y was also de ec ed in neu ons in he e ina (Fig. 1). 2.3. Lack o u in in myeloid cells does no influence he a e o de elopmen al angiogenesis To in es iga e he ole o u in in angiogenesis, we s udied mice deficien o u in gene exp ession in myeloid cells (LysMC e- u (fl/fl) ) and hei espec i e wild- ype li e ma es (LysM WT) du ing de elop- men al e inal angiogenesis and du ing ischemia induced e inal an- giogenesis using oxygen-induced e inopa hy model (OIR). LysMC e- u (fl/fl) mice a e e ile wi hou no ob ious mo phological abno mal- i ies (Co do a e al., 2016), and hei eyes appea no mal upon his o- logical examina ion. As o al deple ion o esiden e inal mac ophages has been shown o slow down he a e o de elopmen al e inal an- giogenesis (Checchin e al., 2006), we fi s explo ed whe he myeloid cell de i ed u in influences he a e o supe ficial ascula plexus o ma ion (de elopmen al angiogenesis) in e inas om P6 LysM WT and LysMC e- u (fl/fl) . In P6 neona al mice, he diame e s o e inal supe ficial ascula plexuses we e simila and no diffe ences in ei he he numbe o b anching poin s o filopodia coun we e de ec ed be- ween LysMC e- u (fl/fl) and LysM WT mice, indica ing ha u in ex- p ession by myeloid cells does no ha e a majo influence on in i o angiogenesis du ing neona al de elopmen (Fig. 2A–E). This finding is in line wi h he abo e epo ed appea ance o u in a he la e s ages o e inal ascula de elopmen (Fig. 1C). 2.4. Mice deficien o u in exp ession in myeloid cells show educed hypoxic e ascula iza ion a e o he e ina The ole o myeloid cell de i ed u in in hypoxia-d i en neo ascu- la iza ion in he e ina was explo ed nex . Fi s , we de e mined whe he eg ession o essels unde hype oxic condi ions in he OIR model is compa able be ween LysMC e- u (fl/fl) and LysM WT mice. A e ex- posu e o 75% oxygen be ween P7 and P12, e inas we e e alua ed a P12. Typical o his model (Smi h e al., 1994; Uusi alo-Jä inen e al., 2007; Vähä upa e al., 2016b), la ge a eas o he cen al ascula ne - wo k we e eg essed wi h only a ew majo essels emaining cen ally in bo h LysM WT and LysMC e- u (fl/fl) mice (Fig. 2F). Quan i a i e analysis o aso-obli e a ed a eas in e ina confi med ha he e inal ascula u e in LysMC e- u (fl/fl) and LysM WT mice was simila ly a - ec ed by hype oxic exposu e (Fig. 2G). Taken oge he , hese da a sugges ha dele ion o u in om myeloid cells has no a significan effec on ascula eg ession in OIR model. On e u n o no moxia, he a ascula cen al e ina becomes hy- poxic and s imula es apid eg ow h o essels (Smi h e al., 1994; Uusi alo-Jä inen e al., 2007). The a e o e inal e ascula iza ion in LysM WT and LysMC e- u (fl/fl) e inas was de e mined by quan i ying he a ascula e inal a ea 5 days a e he mice we e e u ned o no - moxia. The e was a s a is ically significan diffe ence in he a ascula e inal a ea be ween he LysM WT and LysMC e- u (fl/fl) mice a P17 showing ha he a e o e inal e ascula iza ion is subs an ially (30%) educed in mice whe e u in is deple ed om myeloid cells (Fig. 2J). In addi ion o e ascula iza ion o he e ina, he s ong hypoxic s imulus om he cen e o he e ina also d i es abno mal misdi ec ed sp ou ing o blood essels owa ds he i eous a he in e ace be ween he cen ally obli e a ed and pe iphe ally pe used e ina (Smi h e al., 1994; Uusi alo-Jä inen e al., 2007). This pa hological, p e e inal neo ascula iza ion eaches i s maximum 5 days a e e u n o no - moxia (a P17). The e was no s a is ically significan diffe ence in he amoun o p e e inal neo ascula iza ion be ween LysM WT and M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167 161 (cap ion on nex page) M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167 162 LysMC e- u (fl/fl) e inas (Fig. 2K). The esul s indica e ha myeloid cell exp essed u in has a ole in hypoxia-induced e ascula iza ion in e- ina, bu does no influence he pa hological p e e inal neo ascula - iza ion. 3. Discussion The p esen s udy demons a es ha mac ophages associa ed wi h e inal blood essels exp ess u in and he lack o u in in myeloid cells delays hypoxia-d i en angiogenesis in e ina. I has p e iously been shown ha he endo helial specificdeficiency o u in leads o emb y- onal dea h due o ascula de ec s (Roeb oek e al., 1998), and he cul u ed endo helial cells lacking u in canno g ow (Kim e al., 2012). Ou esul s confi m an impo an ole o u in in angiogenesis as we show o he fi s ime ha u in deficiency in myeloid cells leads o impai ed angiogenesis in hypoxia-induced e inal angiogenesis. P e ious s udies ha e shown ha esiden mac ophages, mic oglia, a e needed o p ope angiogenesis in e ina du ing bo h de elopmen al and hypoxia-induced angiogenesis (Checchin e al., 2006). In his s udy, we demons a e ha myeloid-cell specific dele ion o p op o ein con- e ase u in leads o he a enua ion o angiogenesis and educed ascula iza ion a e in OIR model. Among u in's a ge p o eins a e se e al p oangiogenic g ow h ac o s, including VEGF-C which is ex- p essed in mac ophages and has a ole in con olling he b anching o blood essels in he e ina (Sieg ied e al., 2003a; Tammela e al., 2011). VEGF-A, in u n, does no equi e ac i a ing clea age by u in. In e es ingly, a myeloid-cell specific abla ion o VEGF-A exp ession does no change he VEGF le els in he OIR and does no ha e any influence on angiogenesis in he OIR model (Liyanage e al., 2016). Al hough u he s udies conce ning he ole o u in in e inal angio- genesis a e equi ed, ou esul s sugges ha he exp ession o u in in mac ophages may be ela ed o ac i a ion o p oangiogenic g ow h ac o s by u in in he e ina. This is in line wi h p e ious s udies showing simul aneous induc ion o VEGF-C and u in exp ession in diffe en disease models (Kha ib e al., 2010; Lopez de Cicco e al., 2004; Sieg ied e al., 2003a). A majo unc ion o u in is o con ol he bioa ailabili y o an i- inflamma o y TGF-β1(Pesu e al., 2008). As a ma e o ac , bo h u in and TGF-βa e in ol ed in he egula ion o each o he 's ac i i y; u in is needed o he ac i a ion o diffe en TGF-βiso o ms om inac i e p ecu so molecules o ac i e, ma u e o ms (Pesu e al., 2008; Ven u a e al., 2017). The ac i e TGF-β2, in u n, induces u in exp ession (Ven u a e al., 2017). Thus, oge he hey gene a e a sel -sus aining loop o high TGF-βac i i y (Ven u a e al., 2017). TGF-βsignaling is associa ed wi h neo ascula iza ion in many diseases, including neo- ascula ocula diseases (Amin e al., 1994; Bai e al., 2014; Wang e al., 2017). The exp ession o TGF-βis ele a ed in he OIR model, sugges ing ha TGF-βsignaling con ibu es o e inal e ascula iza ion (Yingchuan e al., 2010). A p e ious s udy has shown ha he TGF-β1 bioac i i y is educed in mice deficien in u in exp ession in myeloid cells (Co do a e al., 2016). Thus, ano he plausible explana ion o he educ ion o e ascula iza ion in he e ina o LysMC e- u (fl/fl) mice could be he educed bioa ailabili y o TGF-βdue o lack o ac i a ing u in. Al hough e inal e ascula iza ion a e and he o ma ion o pa- hological p e e inal u s a e in e - ela ed in he OIR model, we iden ified a delayed e ascula iza ion a e in e ina wi hou a sub- sequen inc ease o pa hological p e e inal u o ma ion in LysMC e- u (fl/fl) mice in he OIR model. One explana ion is ha esiden mac- ophages, mic oglia, pe sis in he e ina in esponse o fi e-day long hype oxia despi e blood essels disappea ing comple ely om e ina (Da ies e al., 2006). Once he e ascula iza ion is ini ia ed upon e u n o no moxia, i.e. induc ion o hypoxia in e ina, esiden mic oglial cells espond o hypoxia by sec e ing p oangiogenic g ow h ac o s and di- ec he e ascula iza ion, bu selec i ely only in e ina, whe e hey a e loca ed. This no ion is suppo ed by he ac s ha he mic oglia a e mainly loca ed in he a ascula a eas a e he hype oxia-pe iod and he unc ion o mac ophage-de i ed VEGF-C is o con e he ip cells o s alk cells du ing e inal angiogenesis (Tammela e al., 2011). The lack o influence on p e e inal pa hological e ascula iza ion in he LysMC e- u (fl/fl) mice is a he s iking because we obse ed sig- nifican ly la ge a ascula a eas in LysMC e- u (fl/fl) mice han in WT mice. This, in u n, should heo e ically induce mo e p onounced compensa o y p e e inal pa hological u o ma ion, bu we could no demons a e ha . An explana ion why la ge a ascula e inal a ea does no ansla e in o enhanced p e e inal neo ascula iza ion, may be impai ed p oangiogenic g ow h ac o ac i a ion in LysMC e- u (fl/fl) mice. Taken oge he ou s udy demons a es ha he lack o u in in myeloid cells delays hypoxia-d i en angiogenesis in e ina and im- plica es u in exp essed ou side o he endo helial cells o he egula ion o hypoxia-induced angiogenesis. 4. Me hods 4.1. Mice Monocy e/Mac ophage-specific u in condi ional knockou LysMC e- u (fl/fl) was gene a ed as desc ibed p e iously (Co do a e al., 2016). B iefly, mice bea ing floxed u alleles we e backc ossed six imes wi h C57BL/6 mice. LysMC e mice on C57BL/6 backg ound we e pu chased om Taconic. LysMC e mice we e b ed wi h u (fl/fl) animals o gene a e myeloid-specific u in knockou mice LysMC e- u (fl/fl) . Mice we e housed unde pa hogen- ee s anda d condi ions, b ed and he geno ype was de e mined by PCR. Mice we e ed wi h s anda d labo a o y pelle s and wa e ad libi um. All animal expe imen s we e pe o med acco ding o he ARVO s a emen o he use o animals in oph halmic and ision esea ch in acco dance wi h p o ocols app o ed by he Na ional Animal E hics Commi ee o Finland. 4.2. Oxygen-induced e inopa hy (OIR) model The expe imen s on OIR model we e ca ied ou as desc ibed in de ail p e iously (Smi h e al., 1994; S ahl e al., 2010b; Uusi alo- Jä inen e al., 2007; Vähä upa e al., 2016b). B iefly, neona al mice a P7 and hei nu sing mo he we e exposed o 75% oxygen o 5 days. A P12, he mice we e e u ned o no mal oom ai . Animals we e eu- hanized a P12 o assess he deg ee o ascula eg ession and a P17 o de e mine he a e o e inal e ascula iza ion and p e e inal neo- ascula iza ion. As pos na al weigh gain has been shown o affec ou come in he OIR model, he pups included in he s udy we e weigh - ma ched (S ahl e al., 2010a). Fig. 1. Fu in is exp essed by he endo helial cells and mac ophages in e ina. Using exp ession da a om he FANTOM 5 p ojec , cDNA exp ession da a was ex ac ed o he p ima y u in CAGE o ele an cell ypes (A). Exp ession da a o u in in diffe en e inal cell ypes was ex ac ed om p e iously pe o med mic oa ay exp ession analysis (B). In panels A–B he cell ypes a e so ed by inc easing a e age exp ession om le o igh . Fu in exp ession om e ina was de e mined du ing de elopmen al angiogenesis a P4, P7 and P14 and in OIR model a P14 and P17 om he e ascula ized e inas by IHC and IF using u in specific an ibody. Rep esen a i e images o u in exp ession du ing de elopmen (C) and in he OIR model (D). A ows a e poin ing u in specific s aining a P17 OIR. Scale ba s ep esen s 100 μm. Rep esen a i e con ocal images showing CD31 posi i e endo helial cells ( ed) and u in (g een) (E–F) and F4/80 posi i e mac ophages ( ed) and u in (g een) (G–H) in he e ina o ozen sec ions a e immunofluo escence s aining. Panels E and G a e showing s ainings a 20× magnifica ion and panels F and H a 63× magnifica ion. A ows a e poin ing he endo helial cells o mac ophages ha a e exp essing u in. A owheads a e poin ing neo ascula u s. GCL, ganglion cell laye ; IPL, inne plexi o m laye ; INL, inne nuclea laye ; OPL, ou e plexi o m laye ; ONL, ou e nuclea laye ; IS/OS, pho o ecep o inne /ou e segmen s; RPE, e inal pigmen epi helium. Scale ba s ep esen s 100 μm in E and G and 20 μm in F and H. M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167 163 (cap ion on nex page) M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167 164 4.3. FANTOM analysis o e inal cell ypes The FANTOM p ojec has pe o med exp ession analysis on 1839 samples om 573 p ima y cells, 152 issues, and 250 cell lines in human. Specifically, he da a comes om cap analysis gene exp ession (CAGE) sequencing o cDNA (FANTOM Conso ium e al., 2014). Ex- p ession da a was ex ac ed o he p ima y u in CAGE o ele an cell ypes: endo helial cells, pe icy es, as ocy es, e inal pigmen epi he- lium cells, neu ons, neu ophils, endo helial p ogeni o cells, co neal epi helial cells and monocy e de i ed mac ophages. 4.4. Mic oa ay analysis o e inal cell ypes A mic oa ay exp ession analysis o mouse genes in a ious e inal cell ype g oups was pe o med p e iously and is s o ed as accession E- GEOD-33089 on he A ayExp ess da abase (h ps://www.ebi.ac.uk/ a ayexp ess/expe imen s/E-GEOD-33089). F om he da a we ex- ac ed u in gene exp ession alues o 155 samples ac oss 7 cell ype g oups: amac ine, cone, od, bipola , ho izon al, ganglion, and mic o- glia (Siege e al., 2012). Bo h FANTOM and mic oa ay analyses we e pe o med u ilizing supe compu e esou ces p o ided by CSC–IT Cen e o Science o he Finnish Minis y o Educa ion and Cul u e and he aw da a was p ocessed as p e iously desc ibed in de ail elsewhe e (Ba ke e al., 2017). 4.5. Immunohis ochemis y (IHC) and isolec in GS-IB 4 s aining Fo immunohis ochemis y, he eyes we e fixed wi h 4% PFA and embedded in pa affin ( o IHC), o eshly ozen in OCT embedding compound in liquid ni ogen cooled isopen ane and la e fixed wi h me hanol ( o immunofluo escence). The IHC s ainings we e ca ied ou on 4–6μm hick issue sec ions using he ollowing p ima y an i- bodies: abbi an i- u in, (H-220), (San a C uz Bio echnology, Dallas, TX), a an i-CD31 (BD Pha mingen, San Diego, CA) and a an i-F4/80 (Li e Technologies, Paisley, UK) ollowed by ho se adish pe oxidase (HRP) o fluo escein-conjuga ed seconda y an ibodies. Hema oxylin s aining was used as a coun e s ain. IF samples we e moun ed wi h Vec ashield moun ing medium wi h DAPI (Vec o Labo a o ies, Bu lingame, CA) and images we e aken wi h con ocal mic oscope (Ca l Zeiss LSM 700). As a nega i e con ol, each s aining included sec ions s ained wi hou p ima y an ibody. 4.6. Quan i a i e analysis o angiogenesis Fo he analysis o e inal ascula u e, eyes we e enuclea ed, fixed wi h 4% PFA and e inas dissec ed. Fla moun e inas we e blocked in 10% no mal goa se um o 2 h, incuba ed o e nigh wi h Isolec in GS- IB 4 (1:200, In i ogen, Ca lsbad, CA) Re inas we e imaged ia con ocal mic oscope (Ca l Zeiss LSM 700) and he a e o angiogenesis was de- e mined du ing de elopmen (P6) and in he OIR model as p e iously desc ibed (Conno e al., 2009; S ahl e al., 2010b). B iefly, e inas we e imaged using con ocal mic oscopy (Ca l Zeiss LSM 700) wi h 10 × ob- jec i e by ocusing on he p e e inal neo ascula u s and he unde - lying supe ficial ascula plexus. A eas o ascula obli e a ion and pa hological neo ascula iza ion (neo ascula u s) we e compu ed, in pixels, and di ided by he o al e inal a ea using Adobe Pho oshop CS3. The a e o de elopmen al angiogenesis a P6 was de e mined by measu ing he diame e o ascula u e ia he op ic ne e o he ips o he blood essels. Analysis o essel b anching and numbe o filopodia sp ou s we e quan ified as p e iously desc ibed (Lobo e al., 2007). Each poin whe e h ee capilla y segmen s me was coun ed as one junc ion. B anch poin s om he capilla y plexus om wo a eas (370 μm × 550 μm) pe e ina we e coun ed and esul s a e shown by he a e age coun pe uni a ea. The numbe o filopodia p o usions om he ip cells we e coun ed om he leng h o 500 μm o he sp ou ing ascula on ( wo measu es pe e ina). 4.7. S a is ical analysis S uden 's - es was conduc ed o no mally dis ibu ed da a and nonpa ame ic Mann-Whi ney U es (G aphPad P ism 6.01 and IBM SPSS s a is ics) o non-no mally dis ibu ed da a o es he s a is ical significance o he esul s. P- alues less han 0.05 we e conside ed s a is ically significan . 5. S a emen o au ho con ibu ions M.V, T.J and H. U-J designed he esea ch. M.V and H.B. pe o med he esea ch. M.V, H.B, T.J and H. U-J analyzed he da a. M.P, S.A and Z.M.C p o ided LysMC e- u (fl/fl) mice o he s udy. M.V con ibu ed he geno yped mice li e ma es. M.V, Z.M.C, H.B, T.J and H. U-J w o e he manusc ip . M.V made he figu es. All au ho s e iewed he pape . Conflic o in e es s a emen The au ho s decla e no conflic o in e es . Acknowledgemen s We hank Ma ianne Ka lsbe g o excellen echnical assis ance. The wo k was suppo ed by he Sig id Juselius Founda ion, he Academy o Finland, Päi ikki and Saka i Sohlbe g Founda ion, Ins umen a ium Resea ch Founda ion, Finnish Medical Founda ion, Pi kanmaa Hospi al Dis ic Resea ch Founda ion and he Finnish Cul u al Founda ion, Finnish Diabe ic Resea ch Founda ion, Finnish Eye Founda ion. Re e ences Abcouwe , S.F., 2012. Neu al inflamma ion and he mic oglial esponse in diabe ic e- inopa hy. J. Ocul. Biol. Dis. In o 4, 25–33. Amin, R., Puklin, J.E., F ank, R.N., 1994. G ow h ac o localiza ion in cho oidal neo- ascula memb anes o age- ela ed macula degene a ion. In es . Oph halmol. Vis. Sci. 35, 3178–3188. Bai, Y., Liang, S., Yu, W., Zhao, M., Huang, L., Zhao, M., Li, X., 2014. Semapho in 3A blocks he o ma ion o pa hologic cho oidal neo ascula iza ion induced by ans- o ming g ow h ac o β. Mol. Vis. 20, 1258–1270. Ba ke , H., Aal onen, M., Pan, P., Vähä upa, M., Kaipiainen, P., May, U., P ince, S., Uusi alo-Jä inen, H., Waheed, A., Pas o eko a, S., Sly, W.S., Pa kkila, S., Jä inen, T.A., 2017. Role o ca bonic anhyd ases in skin wound healing. Exp. Mol. Med. 49, e334. Campochia o, P.A., 2015. Molecula pa hogenesis o e inal and cho oidal ascula dis- eases. P og. Re in. Eye Res. 49, 67–81. Checchin, D., Sennlaub, F., Le a asseu , E., Leduc, M., Chem ob, S., 2006. Po en ial ole o mic oglia in e inal blood essel o ma ion. In es . Oph halmol. Vis. Sci. 47, Fig. 2. Fu in deficiency in myeloid cells inhibi s e ascula iza ion a e in he e ina du ing he OIR model. Eyes we e ha es ed om heal hy and LysMC e- u ( fl/fl ) mice a P6 when he de elopmen o he supe ficial ascula plexus has occu ed and in he OIR model a P12 and P17. Re inas we e fixed wi h 4% PFA, fla moun ed and s ained wi h Alexa Fluo conjuga ed Isolec in IB 4 . (A) The a e o ea ly angiogenesis was quan ified in e inal fla moun s by measu ing he leng h o ascula u e ia he op ic ne e. (B) The e was no diffe ence in he leng h o angiogenic ascula u e o supe ficial ascula plexus be ween WT and LysMC e- u ( fl/fl ) e inas a P6. (C) The numbe o b anch poin s (whi e do s) in e inal ascula plexus and filopodia p o usions (yellow do s) om ip cells we e coun ed om P6 e inas. The esul s a e exp essed as he numbe o b anch poin s pe uni a ea (D) and filopodia pe 500 μmo ascula on (E). Bo h ou comes we e simila be ween geno ypes. WT n = 7 and KO n = 6. (F) Rep esen a i e images o e inas igh a e he hype oxic phase o OIR a P12. (G) Quan i a i e analysis o he a ascula a eas (inside he whi e line) shows ha he ascula eg ession is simila in WT and LysMC e- u ( fl/fl ) animals. (WT n = 4, KO n = 7 e inas.) (H) Rep esen a i e OIR model e inas o WT and LysMC e- u ( fl/fl ) mice a P17. (I) The a ascula a eas (yellow) and p e e inal pa hological neo ascula iza ion ( u s, whi e) we e quan ified. (J) Re ascula iza ion a e o e ina is educed by 30% (p = 0.012) in he LysMC e- u ( fl/fl ) mice, while no diffe ences we e de ec ed in he pa hological e ascula iza ion, i.e. in he p e e inal u s (K). (WT n = 26 and KO n = 19 e inas.) Scale ba ep esen s 1 mm in A, F, H and I and 200 μm in C. E o ba s ep esen SDs. M. Vähä upa e al. Expe imen al Eye Resea ch 166 (2018) 160–167 165 3595–3602. Conno , K.M., K ah, N.M., Dennison, R.J., Ade man, C.M., Chen, J., Gue in, K.I., Sapieha, P., S ahl, A., Wille , K.L., Smi h, L.E., 2009. 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