Recombinant Decorin Fusion Protein Attenuates Murine Abdominal Aortic Aneurysm Formation and Rupture

1 SCIENTIFIC REPORTS | 7: 15857 | DOI:10.1038/s41598-017-16194-8 www.na u e.com/scien i ic epo s Recombinan Deco in Fusion P o ein A enua es Mu ine Abdominal Ao ic Aneu ysm Fo ma ion and Rup u e Yue Shen1,2, Vale io Russo1,2, Ma hew R. Zeglinski2, S ephanie L. Selle s1,3, Zhengguo Wu4,5, Came on O am1,2, S ephanie San ac uz1,2, Yulia Me kulo a1,2, Ch is ophe Tu ne 1,2, Kee i Tauh2, Hongyan Zhao1,2, Ta jana Bozin1, Lubos Bohunek1, Haishan Zeng4,5, Michael A. Seidman1, R. Ch is Bleackley6, B uce M. McManus1,10, E kki Ruoslah i7,8, Te o A. H. Jä inen9 & Da id J. G an ille1,2 Deco in (DCN) is a small-leucine ich p o eoglycan ha media es collagen ib illogenesis, o ganiza ion, and ensile s eng h. Ad en i ial DCN is educed in abdominal ao ic aneu ysm (AAA) esul ing in essel wall ins abili y he eby p edisposing he essel o up u e. Recombinan DCN usion p o ein CAR- DCN was enginee ed wi h an ex ended C- e minus comp ised o CAR homing pep ide ha ecognizes in lamed blood essels and pene a es deep in o he essel wall. In he p esen s udy, he ole o sys emically-adminis e ed CAR-DCN in AAA p og ession and up u e was assessed in a mu ine model. Apolipop o ein E knockou (ApoE-KO) mice we e in used wi h angio ensin II (AngII) o 28 days o induce AAA o ma ion. CAR-DCN o ehicle was adminis a ed sys emically un il day 15. Mo ali y due o AAA up u e was signi ican ly educed in CAR-DCN- ea ed mice compa ed o con ols. Al hough he p e alence o AAA was simila be ween ehicle and CAR-DCN g oups, he se e i y o AAA in he CAR-DCN g oup was signi ican ly educed. His ological analysis e ealed ha CAR-DCN ea men signi ican ly inc eased DCN and collagen le els wi hin he ao ic wall as compa ed o ehicle con ols. Taken oge he , hese esul s sugges ha CAR-DCN ea men a enua es he o ma ion and up u e o Ang II-induced AAA in mice by ein o cing he ao ic wall. Abdominal ao ic aneu ysm (AAA) is a common, age- ela ed, li e- h ea ening ascula pa hology ha a ec s app oxima ely 9% o he popula ion o e 65 yea s o age1. AAA is usually asymp oma ic un il up u e, a which poin , mo ali y a es as high as 90% ha e been epo ed2. The e a e no non-in asi e he apeu ics app o ed o p e en ing g ow h and/o up u e. Open su gical o endo ascula epai emain as he only ea men op ions, and a e only ecommended a diame e s abo e 5–5.5 cm a which poin he isk o up u e su passes he isk/ complica ions associa ed wi h su ge y. As such, any pa ien s wi h small AAA expe ience anxie y due o his wai 1Cen e o Hea Lung Inno a ion, S . Paul’s Hospi al, Uni e si y o B i ish Columbia, Vancou e , BC, Canada. 2In e na ional Collabo a ion On Repai Disco e ies (ICORD), Vancou e Coas al Heal h Resea ch Ins i u e and Depa men o Pa hology and Labo a o y Medicine, Uni e si y o B i ish Columbia, Vancou e , BC, Canada. 3Depa men o Radiology, Uni e si y o B i ish Columbia & S . Paul’s Hospi al, Vancou e , BC, Canada. 4Imaging Uni , In eg a i e Oncology Depa men , BC Cance Agency Resea ch Cen e, Vancou e , BC, Canada. 5Pho omedicine Ins i u e, Depa men o De ma ology and Skin Science, Uni e si y o B i ish Columbia & Vancou e Coas al Heal h Resea ch Ins i u e, Vancou e , BC, Canada. 6Depa men o Biochemis y, Uni e si y o Albe a, Edmon on, AB, Canada. 7Cance Resea ch Cen e , San o d-Bu nham P ebys Medical Disco e y Ins i u e, La Jolla, CA, 92037, USA. 8Cen e o Nanomedicine and Depa men o Molecula Cellula and De elopmen al Biology, Uni e si y o Cali o nia, San a Ba ba a, San a Ba ba a, CA, 93106-9610, USA. 9Facul y o Medicine & Li e Sciences, Uni e si y o Tampe e & Depa men o O hopedics & T auma ology, Tampe e Uni e si y Hospi al, Tampe e, Finland. 10PROOF Cen e o Excellence, Uni e si y o B i ish Columbia & P o idence Heal h Ca e, Vancou e , BC, Canada. Co espondence and eques s o ma e ials should be add essed o D.J.G. (email: [email p o ec ed]) Recei ed: 7 Sep embe 2017 Accep ed: 2 No embe 2017 Published: xx xx xxxx OPEN www.na u e.com/scien i ic epo s/ 2 SCIENTIFIC REPORTS | 7: 15857 | DOI:10.1038/s41598-017-16194-8 and wa ch app oach. This has esul ed in an inc eased impe us and need o e ec i e non-in asi e app oaches o p e en o slow AAA p og ession3. The small leucine ich p o eoglycan, deco in (DCN) is ubiqui ously exp essed by nume ous cell ypes, including ib oblas s and smoo h muscle cells. DCN consis s o a 40 kDa co e p o ein ha a aches o a single chond oi in/de ma an sul a e GAG chain. DCN elici s many oles in ex acellula ma ix homeos asis, includ- ing egula ion o collagen ib illogenesis4, collagen deg ada ion5, cell signaling and cell g ow h6–9. Ac ing as an ancho be ween collagen ib ils, DCN p o ides elas ici y and ensile s eng h o collagen ibe s10. Du ing collagen ib illogenesis, DCN is also in ol ed in ib il o ma ion, usion and o ganiza ion4. DCN is also a na u al an agonis o ans o ming g ow h ac o -β (TGF-β), a g ow h ac o associa ed wi h AAA p og ession. Deg ada ion o DCN du ing in lamma ion and emodeling inc eases he TGF-β bioa ailabil- i y, which accele a es ao ic aneu ysm and may a ec in lamma ion11–14. The mu ine se ine p o ease inhibi o , Se pina3n (SA3N), p e en s G anzyme B (GzmB)-media ed DCN deg ada ion and imp o es collagen emode- ling leading o a educed aneu ysm up u e a e and dea h in a mu ine model o AAA15. By sc eening pep ide lib a ies (1.0 × 109) wi h in i o phage display, we ha e iden i ied a ascula homing pep ide o he pu pose o a ge ed deli e y o sys emically adminis e ed he apeu ics o ocal si e wi h speci- ici y16. The ascula homing pep ide CAR (sequence CARSKNKDC) was o iginally iden i ied as i ecognizes angiogenic blood essels and homes o he neo- ascula u e in egene a ing issues16–20. Subsequen wo k es ab- lished ha CAR pep ide also homes in o in lamed essels in condi ions associa ed wi h dis up ed shee s ess/ blood low such as pulmona y a e ial hype ension17,18,20. In addi ion o being a po en homing pep ide, he CAR pep ide is also a cell pene a ing pep ide capable o pene a ing deep in o su ounding pa enchyma and hick a e ial walls as well as deli e ing he pha maceu ical agen s o a ge o gan pa enchyme16–20. We ha e gene - a ed a ecombinan DCN usion p o ein, CAR-DCN, whe e human DCN has been enginee ed wi h an ex ended C- e minus comp ised o he CAR pep ide17. The ancho age o cells a o ded by CAR pep ide in CAR-DCN has subs an ially inc eased i s biological ac i i y and sys emically adminis a ed CAR-DCN accumula ed in he neo- ascula u e- ich wound g anula ion issue in signi ican ly la ge quan i ies han na i e DCN17. In addi ion o ha , CAR-DCN is also subs an ially mo e ac i e han he na i e DCN agains TGF-β17. These ea u es we e asso- cia ed wi h mo e apid wound healing and supp essed sca o ma ion when compa ed o imp o emen ob ained by na i e DCN17. AAA p og ession is associa ed wi h ongoing in lamma ion in conce wi h inc eased angiogenesis wi hin he essel wall21. Inc eased angiogenesis and exp ession o angiogenic, in lamma o y cy okines a e obse ed a he si e o aneu ysm up u e22,23. Based on ou p e ious s udies pe aining o he ole o DCN in he de elopmen o AAA15, combined wi h he neo- ascula u e- and in lamma o y-homing cha ac e is ics o he CAR-DCN pep ide p omp ed us o in es iga e whe he sys emic adminis a ion o CAR-DCN could a ec he onse and p og ession o AAA. In he p esen s udy, CAR-DCN ea men was assessed using an angio ensin II (Ang II)-induced AAA model. We hypo hesized ha sys emically-adminis e ed CAR-DCN would a enua e AAA p og ession and up- u e, and inc ease su i al. Resul s CAR-DCN T ea men Inc eases 28-day Su i al and Reduces Se e i y o Ang II-induced AAA. CAR-DCN- ea ed, Ang II-in used ApoE-KO mice exhibi ed a signi ican inc ease in 28-day su i al (92.8%, n = 14; s 60%, n = 15; P = 0.035) in compa ison o ehicle- ea ed con ols (Fig.1A). Nec opsy was pe o med on all mice ha died be o e he 28-day ime poin o de e mine cause o dea h. All cases o p ema u e dea h exhibi ed signs o exsanguina ion in he abdominal ca i y indica i e o AAA up u e. To ollow AAA p og es- sion, all mice we e examined by ul asound a h ee ime poin s (day 0 p io o pump implan a ion, and day 7 and day 21 pos -implan a ion). AAA was de ined as a >50% enla gemen o he maximum ao ic diame e in sham ApoE-KO mice, in line wi h he cu en clinical de ini ion24. The CAR-DCN ea ed g oup exhibi ed a smalle a e age maximum ao ic diame e a bo h day 7 (1.39 ± 0.07, n = 13; s 1.56 ± 0.11, n = 11) and day 21 (1.69 ± 0.10, n = 13; s 1.93 ± 0.21, n = 9) when compa ed wi h he ehicle con ol g oup (Fig.1B), howe e he di e ence did no each s a is ical signi icance. The onse o AAA was simila be ween CAR-D CN and ehi- cle g oups (Fig.1C). All ao as we e ha es ed and his ologically assessed o aneu ysm. Based on p e iously published c i e ia25, aneu ysms we e ca ego ized in o h ee classes: small AAA, la ge AAA and up u ed AAA (Fig.2A). Among all mice ha de eloped AAA, g ea e han 50% o ehicle- ea ed mice exhibi ed an aneu ysmal up u e (54.5%, n = 6/11) compa ed o only 10% o CAR-DCN- ea ed mice (10%, n = 1/10) (Fig.2B). Al hough he p e alence o la ge bu non- up u ed aneu ysm was simila be ween ehicle and CAR-DCN g oups (27.2%, n = 3/11; s 20%, n = 2/10), he majo i y o aneu ysms in CAR-DCN g oup we e small AAAs (70%, n = 7/10 s. 18% in ehicle con ol, n = 2/11) (Fig.2B). The p e alence o aneu ysm was analyzed using a chi-squa e es and a s a is ically signi ican di e ence was obse ed be ween he ehicle con ol g oup and CAR-DCN g oup (P = 0.038). CAR-DCN T ea men Inc eases Ad en i ial Collagen O ganiza ion. Ad en i ial DCN was ma k- edly highe in he CAR-DCN g oup as compa ed o ehicle con ols (Fig.3A). Ad en i ial collagen was assessed using pic osi ius ed s aining, collagen ibe s om he ehicle con ol g oup s ained g een and yellow, indica - ing a weake and imma u e collagen s uc u e ha was consis en wi h p e ious s udies15. Con e sely, collagen ibe s om he CAR-DCN g oup appea ed o be mos ly o ange and ed, sugges ing a mo e ma u e, o ganized collagen s uc u e (Fig.3B). These indings we e con i med using second ha monic gene a ion (SHG) collagen analysis (Fig.3C). To con i m whe he CAR pep ide homes o he ad en i ia in AAA, a pep ide homing s udy was pe o med using luo escen -labelled pep ides. Bo h CAR and mu an CAR pep ide we e i. .-adminis e ed in o hese animals o de e mine pep ide homing o AAA. The s ong g een au o luo escence o he abdominal ao a and he pene a ion o CAR pep ide o pa enchyme make i di icul o accu a ely in e p e he da a o di ec www.na u e.com/scien i ic epo s/ 3 SCIENTIFIC REPORTS | 7: 15857 | DOI:10.1038/s41598-017-16194-8 FAM imaging. Fo his eason, and o p ese e he his ologic ea u es, we used IHC s aining wi h an i- luo escein an ibodies o de ec he FAM-labeled pep ides. S ong accumula ion o CAR was obse ed in he ao ic ad en i ia bo h in blood essels and he su ounding pa enchyme (Supplemen al Figu eS1). CAR-DCN T ea men Does No A ec Medial Dis up ion. Al hough he CAR-DCN g oup also showed an inc ease in he a e age ad en i ial hickness as compa ed o he ehicle con ol g oup (55.8 µm in CAR-DCN g oup s 39.0 µm in ehicle con ol g oup), he di e ence was no s a is ically signi ican (Fig.4A). The medial hickness o CAR-DCN and ehicle g oups was compa able (Fig.4A). Se e e elas ic ibe agmen- a ion was obse ed in bo h CAR-DCN and ehicle g oups and he p e alence o medial dis up ion be ween hese wo g oups we e simila , indica ing ha CAR-DCN ea men did no p e en medial dis up ion (Fig.4B). Fib illin-1 le els in he unica media we e also simila be ween ehicle and CAR-DCN g oups (Fig.4C). GzmB was de ec ed in he ao as o mice ha exhibi ed AAA, and he e was no di e ence on he le el o GzmB s aining be ween ehicle con ol and CAR-DCN g oups (Supplemen al Figu eS2A). To examine whe he CAR-DCN is subjec o GzmB-media ed clea age, a GzmB-CAR-DCN clea age assay was pe o med. GzmB educed ull-leng h CAR-DCN le els, which was abolished by inhibi ion o GzmB wi h SA3N (Supplemen al Figu eS2B). Figu e 1. CAR-DCN ea men imp o es 28-day su i al in Ang II-in used ApoE-KO mice. (A) 28-day su i al a e in Ang II in used ApoE-KO mice ea ed wi h saline (60%, n = 15) o CAR-DCN (92.8%, n = 14). P = 0.035 by Log- ank (Man el-Cox) es . (B) Sca e plo o maximum diame e o abdominal ao a in Ang II-in used ApoE-KO mice ea ed wi h ehicle o CAR-DCN. Solid line indica es he mean alue o each g oup. Dash line indica es he AAA h eshold (>50% enla gemen o maximum ao ic diame e in sham ApoE-KO mice). (C) AAA p e alence in Ang II-in used ApoE-KO mice ea ed wi h ehicle o CAR-DCN a day 7 and day 28, AAA is de e mined by ul asonic measu emen s. www.na u e.com/scien i ic epo s/ 4 SCIENTIFIC REPORTS | 7: 15857 | DOI:10.1038/s41598-017-16194-8 Discussion Ao ic aneu ysm is a leading cause o dea h in No h Ame ica26. Smoking, male gende , aging and amily his o y a e he majo isk ac o s o AAA26,27. The isk o AAA inc eases d ama ically a e 60 yea s o age28. Cu en ly, he only ea men op ions a ailable a e elec i e open su gical epai , which is associa ed wi h a 5.6% ope a i e mo ali y, o endo ascula epai , which is associa ed wi h inc eased isk o pos -su ge y up u e. To da e, no app o ed, non-su gical in e en ions a e a ailable28. The p esen s udy sugges s ha sys emic adminis a ion o CAR-DCN, a a ge issue-speci ic non-su gical in e en ion, can educe AAA se e i y and up u e by inc easing ad en i ial collagen o ganiza ion. P e ious s udies ha e demons a ed ha inhibi ion o GzmB-media ed ad en i ial DCN deg ada ion leads o inc eased collagen densi y and educed aneu ysm up u e15. In he p esen s udy, we es ed an al e na i e app oach by compensa ing he loss o DCN in AAA h ough exogenous DCN supplemen a ion. A 15-day eg- imen was de e mined based on p e ious s udies in which he as majo i y o up u es in Ang II-induced AAA occu ed wi hin he i s wo weeks o Ang II in usion15,25,29,30. CAR pep ide is a cell/ issue pene a ing pep ide ha has also been shown o speci ically accumula e in he ad en i ia in a mu ine model o pulmona y a e ial hype ension18,19. In he p esen s udy, ad en i ial DCN con en was signi ican ly inc eased. Addi ionally, colla- gen hick bundle o ma ion was g ea e wi h CAR-DCN ea men as compa ison o ehicle con ols. Al hough CAR-DCN ea men did no a ec aneu ysm onse , aneu ysm se e i y was signi ican ly a enua ed based on 28-day su i al, ul asound measu emen s and pa hology examina ion. One possible limi a ion o CAR-DCN ea men compa ed o p e ious AAA s udies whe e GzmB was de icien o inhibi ed15,25 is ha CAR-DCN ea men did no p e en medial dis up ion o change GzmB le els. Ou in i o da a u he showed ha CAR-DCN is suscep ible o GzmB-media ed clea age. Du ing aneu ysm p og ession, GzmB clea es many ex acellula ma ix p o eins including DCN and ib illin-1 bo h o which play pi o al oles in main- aining essel wall s abili y15,25. Despi e a signi ican a enua ion o AAA p og ession, CAR-DCN ea men did no appea o a ec o he ma ix p o eins such as ib illin-1 ha a e subjec o GzmB-media ed p o eolysis. This aises a possibili y o u he e-enginee ing CAR-DCN whe eby he GzmB clea age si e is mu a ed o be esis an o clea age. The majo i y o human AAA samples a e collec ed om au opsy o elec i e su ge y, a which poin he s age o aneu ysm is conside ed o be ad anced, la e s age lesions. Cu en ly, he e a e se e al animal models o ao ic aneu ysm a ailable o which he Ang II AAA model is he mos widely used. These animal models o Figu e 2. CAR-DCN ea men educes AAA se e i y in Ang II-in used ApoE-KO mice. (A) Rep esen a i e g oss pa hology and mo phology o di e en aneu ysm class. Yellow a ows deno e ao ic aneu ysm. Ao as we e s ained wi h H&E o assess mo phology. Scale ba : 1 mm. (B) P e alence o aneu ysm class in Ang II- in used ApoE-KO mice ea ed wi h ehicle (n = 15) o CAR-DCN (n = 14), P = 0.038 by chi-squa e es . www.na u e.com/scien i ic epo s/ 5 SCIENTIFIC REPORTS | 7: 15857 | DOI:10.1038/s41598-017-16194-8 AAA eplica e many o he cellula and biochemical cha ac e is ics o he human disease, including a ch onic in lamma o y esponse and ex ensi e ex acellula ma ix deg ada ion and a e se e e enough o be e en ually le hal29,31,32. Al hough animal models o AAA display ce ain ea u es ha a e dis inc om wha is seen in human AAA samples, hey allow us o in es iga e he ini ial pa hological changes o he disease and examine he ea ly in e en ions. Simila o wha we ha e seen in he pas 15 and in he p esen s udy, a ecen s udy om Ueda e al. con i med ha he le el o DCN dec eased a he ea ly s age o disease. In addi ion, hey u he showed ha implan a ion o exogenous DCN con aining Gel oam pa ches in he pe iao ic space p e en ed he de elopmen o AAA33. In he p esen s udy, we used a no el p o ein CAR-DCN, which is mo e ac i e han na i e DCN agains TGF-β, an impo an playe in abdominal aneu ysm17. Fu he mo e, as opposed o p e ious s udies33, in which su gical implan a ion o gel oam pa ches con aining DCN was u ilized o localize DCN o he pe iao ic space, a non-in asi e, mo e clinically- iable app oach, i.e. sys emic adminis a ion o CAR-DCN, was applied. As such, gi en simila esul s we e obse ed, ou s udies suppo he sys emic adminis a ion o CAR-DCN as a clinically iable, non-su gical in e en ion o ea ly/small AAA ea men . Figu e 3. CAR-DCN ea men inc eases ad en i ial DCN and ad en i ial collagen o ganiza ion. Rep esen a i e (A) deco in and (B) pic osi ius ed s aining (le ) and quan i ica ion o (A) deco in and (B) pic osi ius ed s aining in ensi y ( igh ) in abdominal ao as om o su i ing ApoE-KO mice in di e en ea men g oups (n ≥ 6 pe g oup). M, media; Ad, ad en i ia; Scale ba : 200 μm. (C) Rep esen a i e SHG imaging (le ) and quan i ica ion o SHG signal in ensi y ( igh ) in ad en i ia o abdominal ao as om su i ing ApoE-KO mice in di e en ea men g oups (n ≥ 6 pe g oup). Scale ba : 20 μm. Resul s a e exp essed as box-and-whiske plo , *P < 0.05, **P < 0.01 by S uden - es . www.na u e.com/scien i ic epo s/ 6 SCIENTIFIC REPORTS | 7: 15857 | DOI:10.1038/s41598-017-16194-8 Figu e 4. CAR-DCN ea men does no p o ec agains medial dis up ion. (A) Quan i ica ion o ad en i ial and medial hickness in abdominal ao as o ApoE-KO mice in di e en ea men g oups (n ≥ 6 pe g oup). (B) Rep esen a i e Mo a ’s pen ach ome s aining (le ) and p e alence o medial dis up ion ( igh ) in abdominal ao as om su i ing ApoE-KO mice in di e en ea men g oups. A ows indica e medial dis up ion. M, media; Ad, ad en i ia; Scale ba : 200 μm. (C) Rep esen a i e ib illin-1 s aining (le ) and quan i ica ion o ib illin-1 s aining in ensi y ( igh ) in unica media om o su i ing ApoE-KO mice in di e en ea men g oups (n ≥ 6 pe g oup). M, media; Ad, ad en i ia; Scale ba : 200 μm. Resul s a e exp essed as box-and-whiske plo , ns = no signi ican . www.na u e.com/scien i ic epo s/ 7 SCIENTIFIC REPORTS | 7: 15857 | DOI:10.1038/s41598-017-16194-8 Taken oge he , he cu en s udy demons a es he u ili y o a no el he apeu ic app oach o a enua e AAA p og ession h ough sys emic adminis a ion o CAR-DCN. In conclusion, ou indings suppo he u u e de el- opmen o an AAA he apy in ol ing sys emic adminis a ion o CAR-DCN o delay and/o p e en aneu ysmal up u e. Ma e ials and Me hods Mice. Apolipop o ein E knockou (ApoE-KO) male mice (C57Bl/6 backg ound, S ock No. 002052) we e ob ained om Jackson Labo a o ies, Ba Ha bo , ME, USA. All mice we e housed a The Gene ic Enginee ed Models acili y a S . Paul’s Hospi al, Uni e si y o B i ish Columbia. All p ocedu es we e pe o med in acco d- ance wi h he guidelines o animal expe imen a ion app o ed by he Animal Expe imen a ion Commi ee o he Uni e si y o B i ish Columbia. Angio ensin II–Induced AAA. AAA was induced by Ang II in usion, as p e iously desc ibed15. Recombinan P o ein P oduc ion. Recombinan CAR-DCN was exp essed in 293-F cells using he F eeS yle 293 exp ession sys em as desc ibed p e iously16. B ie ly, a pcDNA3.1/myc-his-C plasmid encoding o CAR-DCN was mixed wi h Op i-MEM I media and 293 ec in ans ec ion eagen . The DNA:293 ec in solu ion was added o 293-F cells and he cells we e cul u ed o 48 h. Recombinan p o ein was isola ed om he media u ilizing poly-his idine ag binding on Ni-NTA aga ose beads (Qiagen) using 5 mL o beads pe 500 mL o media. A e an o e nigh incuba ion a 4 °C, he beads we e washed wi h PBS, and CAR-DCN was elu ed wi h PBS con- aining 300 mM imidazole, dialyzed agains PBS, and s o ed a −80 °C. CAR-DCN T ea men . Ang II-in used ApoE-KO mice we e gi en a ail ein injec ion o ehicle con ol (PBS) o CAR-DCN (40 μg/100 μL PBS pe injec ion) e e y hi d day un il day 15. In ape i oneal injec ion o CAR-DCN o ehicle con ol was pe o med once a day be ween ail ein injec ions. The CAR-DCN ea men egimen is shown in Supplemen al Figu eS2. The dose o he ea men was selec ed on he basis o p e ious s udies17. In Vi o Ul asound Measu emen . Abdominal ao as o he mice we e isualized using a VisualSonics Ve o 2100 High-Resolu ion Imaging Sys em wi h a 40-MHz equency ansduce (Fuji ilm VisualSonics, To on o, ON, Canada) be o e he implan a ion and a day 7 and day 21 pos -implan a ion, as p e iously desc ibed34. Tissue Collec ion and P epa a ion. Fou weeks ollowing implan a ion, issues om su i ing mice we e ha es ed as p e iously desc ibed15. Ao ic segmen s we e isola ed om he abdominal ao a immedia ely abo e he enal a e ies and s o ed in 10% o malin o e nigh be o e embedding in pa a in and sec ioning. Nec opsies we e pe o med on all mice ha died be o e he 4-week ime poin o de e mine he cause o dea h. His ological Analysis. Abdominal ao ic sec ions we e s ained wi h Mo a ’s pen ach ome and pic osi ius ed. Immunohis ochemis y was pe o med using abbi an i-mouse G anzyme B (Abcam, Camb idge, MA, USA), abbi an i-Fib illin-1 (Abcam), abbi an i- luo escen (The moFishe , S . Louis, MO, uSA) and goa an i-mouse deco in (R&D Sys ems, Minneapolis, MN, USA) as desc ibed p e iously15. His ological e alua ion o se e i y o disease was pe o med independen ly by wo expe imen al pa hologis s who we e blinded o he expe imen al condi ions. Second Ha monic Gene a ion Mic oscopy and Collagen Analysis. Collagen in he essel wall was isualized using a cus omized ideo a e mul imodali y mul ipho on mic oscopy sys em35. Image acquisi ion was ca ied ou a 15 ames pe second wi h a esolu ion o 512 by 512 pixels. The lase sou ce was an 80 MHz Ti:Sappi e em osecond lase (Chameleon, Cohe en Inc., San a Cla a, CA, USA) wi h a wa eleng h uning ange o 700 nm-950 nm. The as imaging speed was ealized by using an 8 kHz esonance scanne o he as axis and a gal anome e scanne o he slow axis. A 60X (NA = 1.0) wa e -imme sion objec i e (LUMPLFLN60X/W, Olympus Canada, Ma kham, ON, Canada) was used o ocus he lase ligh in o he sample. The second ha - monic signal was collec ed in he epi-di ec ion by he same objec i e and was hen e lec ed by a dich oic mi - o (FF665-Di02–25 × 36, Sem ock, Inc., Roches e , NY, USA) and ocused in o a pho omul iplie ubes (PMT, H9433MOD-03, Hamama su Co p., B idgewa e , NJ, USA). A band pass il e (FF01–390/40–25, Sem ock, Inc.) was loca ed in on o he PMT wi h a ansmission ange om 370 nm o 410 nm o SHG de ec ion wi h an exci a ion wa eleng h o 800 nm. Acqui ed images we e a e aged e e y 10 ames o imp o e he signal o noise a io. The o al SHG signal in ensi y alues we e quan i ied by ImageJ 1.5i. CAR-DCN Clea age Assay. CAR-DCN (5 µg) was incuba ed wi h 200 nM o human GzmB (Be yllium, Bos on, MA, USA) o e nigh a 37 °C in a wa e ba h in diges ion bu e (100 mM HEPES pH 7.5, 0.20% w/ CHAPS, 10 mM DTT). To demons a e he unc ionali y o GzmB o clea e CAR-DCN, we p e-incuba ed 200 nM o GzmB (Be yllium) wi h 600 nM o Se pin A3N (SA3N), (a gene ous gi om D . Ch is R. Bleackley, Uni e si y o Albe a, Edmon on, AB, Canada) o 60 minu es a 37 °C in a wa e ba h. A e p e-incuba ion, 5.0 µg o CAR-DCN was added o each eac ion and incuba ed o e nigh a 37 °C in a wa e ba h. A e incuba ion, p o- eins we e dena u ed and sepa a ed on a 10% SDS- polyac ylamide gel. The gel was s ained by SimplyBlue Sa e S ain (In i ogen, Bu ling on, ON, Canada) and imaged using he LICOR Odyssey Fc (LI-COR Bio echnology, Lincoln, NE, USA) unde he 600 channel wi h a 2 minu e acquisi ion cycle. Image was hen pseudocolou ed o black on whi e o easie isualiza ion. Fib onec in (92784, Abcam) was also used in his assay as posi i e con ol. www.na u e.com/scien i ic epo s/ 8 SCIENTIFIC REPORTS | 7: 15857 | DOI:10.1038/s41598-017-16194-8 Pep ide Ta ge ing S udy. The ollowing pep ides labeled wi h FITC o 5-ca boxy luo escein (FAM) we e used o he ao a a ge ing s udies: CAR, CARSKNKDC and CAR mu an (con ol pep ide), CAQSNNKDC. Pep ides we e dissol ed in PBS a concen a ions o 0.5 mg/mL. ApoE-KO mice we e injec ed wi h pep ide solu- ion h ough he ail ein (3 mg/kg) h ee days a e AngII in usion. Two hou s a e injec ion, he mice we e pe used wi h PBS con aining 1% bo ine se um albumin while unde deep anes hesia, and issues we e ixed by sys emic pe usion wi h 10% bu e ed o malin. The ao as we e excised and ixed o an addi ional 24 hou s and p ocessed o an i- luo escein immunohis ochemis y (IHC) analysis as desc ibed p e iously18,19. S a is ical Analysis. Quan i a i e alues a e exp essed as mean ± SEM. S a is ical analysis was pe o med using G aphPad P ism e sion 5.01 (G aphPad So wa e, San Diego, CA, USA). Su i al cu es we e assessed using Log- ank es o end and Log- ank/Man el-Cox analysis. P e alence o AAA class was assessed by chi-squa e es . Deco in in ensi y, collagen densi y and SHG signal in ensi y was assessed by unpai ed S uden ’s - es . Fo all es s, signi ican di e ence we e se a P < 0.05. Da a A ailabili y. All da a gene a ed o analysed du ing his s udy a e included in his published a icle (and i s Supplemen a y In o ma ion iles). Re e ences 1. Thompson, R. W. De ec ion and managemen o small ao ic aneu ysms. The New England jou nal o medicine 346, 1484–1486 (2002). 2. Ken , K. C. Clinical p ac ice. Abdominal ao ic aneu ysms. The New England jou nal o medicine 371, 2101–2108 (2014). 3. Bax e , B. T., Te in, M. C. & Dalman, R. L. Medical managemen o small abdominal ao ic aneu ysms. Ci cula ion 117, 1883–1889 (2008). 4. Reed, C. C. & Iozzo, R. V. The ole o deco in in collagen ib illogenesis and skin homeos asis. Glycoconjuga e jou nal 19, 249–255 (2002). 5. Bhide, V. M. e al. Collagen phagocy osis by ib oblas s is egula ed by deco in. The Jou nal o biological chemis y 280, 23103–23113 (2005). 6. Kinsella, M. G., B essle , S. L. & Wigh , T. N. The egula ed syn hesis o e sican, deco in, and biglycan: ex acellula ma ix p o eoglycans ha in luence cellula pheno ype. C i ical e iews in euka yo ic gene exp ession 14, 203–234 (2004). 7. Fische , J. W., Kinsella, M. G., Le kau, B., Clowes, A. W. & Wigh , T. N. Re o i al o e exp ession o deco in di e en ially a ec s he esponse o a e ial smoo h muscle cells o g ow h ac o s. A e ioscle osis, h ombosis, and ascula biology 21, 777–784 (2001). 8. Mosca ello, D. K. e al. Deco in supp esses umo cell g ow h by ac i a ing he epide mal g ow h ac o ecep o . The Jou nal o clinical in es iga ion 101, 406–412 (1998). 9. Seidle , D. G. e al. Deco in p o ein co e inhibi s in i o cance g ow h and me abolism by hinde ing epide mal g ow h ac o ecep o unc ion and igge ing apop osis ia caspase-3 ac i a ion. The Jou nal o biological chemis y 281, 26408–26418 (2006). 10. Pins, G. D., Ch is iansen, D. L., Pa el, R. & Sil e , F. H. Sel -assembly o collagen ibe s. In luence o ib illa alignmen and deco in on mechanical p ope ies. Biophysical jou nal 73, 2164–2172 (1997). 11. Imai, K., Hi ama su, A., Fukushima, D., Pie schbache , M. D. & Okada, Y. Deg ada ion o deco in by ma ix me allop o einases: iden i ica ion o he clea age si es, kine ic analyses and ans o ming g ow h ac o -be a1 elease. The Biochemical jou nal 322(P 3), 809–814 (1997). 12. Boi in, W. A. e al. G anzyme B clea es deco in, biglycan and soluble be aglycan, eleasing ac i e ans o ming g ow h ac o -be a1. PloS one 7, e33163 (2012). 13. A H Zen, A. e al. E ec o adeno i us-media ed o e exp ession o deco in on me allop o einases, issue inhibi o s o me allop o einases and cy okines sec e ion by human gingi al ib oblas s. Ma ix biology: jou nal o he In e na ional Socie y o Ma ix Biology 22, 251–258 (2003). 14. Jä inen, T. A. & P ince, S. Deco in: A G ow h Fac o An agonis o Tumo G ow h Inhibi ion. BioMed esea ch in e na ional 2015, 654765 (2015). 15. Ang, L. S. e al. Se pina3n a enua es g anzyme B-media ed deco in clea age and up u e in a mu ine model o ao ic aneu ysm. Cell dea h & disease 2, e209 (2011). 16. Jä inen, T. A. & Ruoslah i, E. Molecula changes in he ascula u e o inju ed issues. The Ame ican jou nal o pa hology 171, 702–711 (2007). 17. Jä inen, T. A. & Ruoslah i, E. Ta ge -seeking an i ib o ic compound enhances wound healing and supp esses sca o ma ion in mice. P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o Ame ica 107, 21671–21676 (2010). 18. U akami, T. e al. Pep ide-di ec ed highly selec i e a ge ing o pulmona y a e ial hype ension. The Ame ican jou nal o pa hology 178, 2489–2495 (2011). 19. Toba, M. e al. A no el ascula homing pep ide s a egy o selec i ely enhance pulmona y d ug e icacy in pulmona y a e ial hype ension. The Ame ican jou nal o pa hology 184, 369–375 (2014). 20. Jä inen, T. A. e al. Sys emic a ge ed deli e y o mul i- unc ional ecombinan p o eins and nanopa icles in egene a i e medicine. ACS bioma e ials in science & enginee ing 3, 1273–1282 (2017). 21. Thompson, M. M., Jones, L., Nasim, A., Saye s, R. D. & Bell, P. R. Angiogenesis in abdominal ao ic aneu ysms. Eu opean jou nal o ascula and endo ascula su ge y: he o icial jou nal o he Eu opean Socie y o Vascula Su ge y 11, 464–469 (1996). 22. Choke, E. e al. Gene exp ession p o ile o abdominal ao ic aneu ysm up u e. Annals o he New Yo k Academy o Sciences 1085, 311–314 (2006). 23. Choke, E. e al. Abdominal ao ic aneu ysm up u e is associa ed wi h inc eased medial neo ascula iza ion and o e exp ession o p oangiogenic cy okines. A e ioscle osis, h ombosis, and ascula biology 26, 2077–2082 (2006). 24. Johns on, K. W. e al. Sugges ed s anda ds o epo ing on a e ial aneu ysms. Subcommi ee on Repo ing S anda ds o A e ial Aneu ysms, Ad Hoc Commi ee on Repo ing S anda ds, Socie y o Vascula Su ge y and No h Ame ican Chap e , In e na ional Socie y o Ca dio ascula Su ge y. Jou nal o ascula su ge y 13, 452–458 (1991). 25. Chambe lain, C. M. e al. Pe o in-independen ex acellula g anzyme B ac i i y con ibu es o abdominal ao ic aneu ysm. The Ame ican jou nal o pa hology 176, 1038–1049 (2010). 26. Agga wal, S., Qama , A., Sha ma, V. & Sha ma, A. Abdominal ao ic aneu ysm: A comp ehensi e e iew. Expe imen al and clinical ca diology 16, 11–15 (2011). 27. Hendel, A., Ang, L. S. & G an ille, D. J. In lammaging and p o eases in abdominal ao ic aneu ysm. Cu en ascula pha macology 13, 95–110 (2015). 28. Powell, J. T. & G eenhalgh, R. M. Clinical p ac ice. Small abdominal ao ic aneu ysms. The New England jou nal o medicine 348, 1895–1901 (2003). 29. Golledge, J., Mulle , J., Daughe y, A. & No man, P. Abdominal ao ic aneu ysm: pa hogenesis and implica ions o managemen . A e ioscle osis, h ombosis, and ascula biology 26, 2605–2613 (2006). www.na u e.com/scien i ic epo s/ 9 SCIENTIFIC REPORTS | 7: 15857 | DOI:10.1038/s41598-017-16194-8 30. Cao, R. Y., Amand, T., Fo d, M. D., Piomelli, U. & Funk, C. D. The Mu ine Angio ensin II-Induced Abdominal Ao ic Aneu ysm Model: Rup u e Risk and In lamma o y P og ession Pa e ns. F on ie s in pha macology 1, 9 (2010). 31. Daughe y, A. & Cassis, L. A. Mouse models o abdominal ao ic aneu ysms. A e ioscle osis, h ombosis, and ascula biology 24, 429–434 (2004). 32. Kui aniemi, H., Rye , E. J., Elmo e, J. R. & T omp, G. Unde s anding he pa hogenesis o abdominal ao ic aneu ysms. Expe e iew o ca dio ascula he apy 13, 975–987 (2015). 33. Ueda, K. e al. Possible dual ole o deco in in abdominal ao ic aneu ysm. PloS one 10, e0120689 (2015). 34. Shen, Y. e al. G anzyme B De iciency P o ec s agains Angio ensin II-Induced Ca diac Fib osis. The Ame ican jou nal o pa hology 186, 87–100 (2016). 35. Lee, A. M. e al. In i o ideo a e mul ipho on mic oscopy imaging o human skin. Op ics le e s 36, 2865–2867 (2011). Acknowledgemen s We hank Am i Sam a and D . Sa ah J. Williams o echnical suppo . Suppo ed by he Canadian Ins i u es o Heal h Resea ch (CIHR) g an (D.J.G., R.C.B. and H.Z.), he CIHR Pos doc o al Fellowship (Y.S., M.Z., C.T.), he F ede ick Ban ing and Cha les Bes Canada G adua e Schola ship (Y.M., K.T.), he Anne and John B own Fellowship in Diabe es and Obesi y Rela ed Resea ch (S.S.), he Academy o Finland, Päi ikki and Saka i Sohlbe g Founda ion, Ins umen a ium Resea ch Founda ion, Tampe e Tube culosis Founda ion, Pi kanmaa Hospi al Dis ic Resea ch Founda ion (T.A.H.J.). Au ho Con ibu ions Y.S., T.A.H.J., and D.J.G. concei ed and designed he expe imen s; Y.S., V.R., M.R.Z., Z.W., C.O., S.S., Y.M., H.Z., T.B., and L.B. pe o med he expe imen s; Y.S., V.R., M.R.Z., Z.W., S.L.S., B.M.M., and M.A.S. analyzed he da a. Y.S., M.R.Z., C.T., K.T., H.Z., R.C.B., B.M.M., E.R., T.A.H.J., and D.J.G. w o e and p epa ed he manusc ip . Addi ional In o ma ion Supplemen a y in o ma ion accompanies his pape a h ps://doi.o g/10.1038/s41598-017-16194-8. Compe ing In e es s: The au ho s decla e ha hey ha e no compe ing in e es s. Publishe 's no e: Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C e- a i e Commons license, and indica e i changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons license, unless indica ed o he wise in a c edi line o he ma e ial. I ma e ial is no included in he a icle’s C ea i e Commons license and you in ended use is no pe - mi ed by s a u o y egula ion o exceeds he pe mi ed use, you will need o ob ain pe mission di ec ly om he copy igh holde . 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