Vulnerability of progeroid smooth muscle cells to biomechanical forces is mediated by MMP13

ARTICLE Vulne abili y o p oge oid smoo h muscle cells o biomechanical o ces is media ed by MMP13 Pa icia R. Pi ez 1,2, Luís Es onca1,2, Luís Miguel Mon ei o 1, Guillem Colell3, Helena Vazão1, Deolinda San inha 1,2, Ka im Ha hou i 4, Daniel Tho n on5, Clai e Na a o 4,6, Anne-Lau e Egesipe7, Tânia Ca alho 8, Rod igo L. Dos San os9, Nicolas Lé y4,10, James C. Smi h 11, João Ped o de Magalhães1,5, Alessand o O i 12, And eia Be na do 11, Annachia a De Sand e-Gio annoli 4,10,13, Xa ie Nissan 7, Anna Rosell3& Lino Fe ei a 1,2✉ Hu chinson-Gil o d P oge ia Synd ome (HGPS) is a p ema u e aging disease in child en ha leads o ea ly dea h. Smoo h muscle cells (SMCs) a e he mos a ec ed cells in HGPS indi iduals, al hough he eason o such ulne abili y emains poo ly unde s ood. In his wo k, we de elop a mic ofluidic chip o med by HGPS-SMCs gene a ed om induced plu - ipo en s em cells (iPSCs), o s udy hei ulne abili y o flow shea s ess. HGPS-iPSC SMCs cul u ed unde a e ial flow condi ions de ach om he chip a e a ew days o cul u e; his p ocess is media ed by he up egula ion o me allop o ease 13 (MMP13). Impo an ly, double-mu an LmnaG609G/G609GMmp13−/−mice o LmnaG609G/G609GMmp13+/+mice ea- ed wi h a MMP inhibi o show lowe SMC loss in he ao ic a ch han con ols. MMP13 up egula ion appea s o be media ed, a leas in pa , by he up egula ion o glycocalyx. Ou HGPS-SMCs chip ep esen s a pla o m o de eloping ea men s o HGPS indi iduals ha may complemen p e ious p e-clinical and clinical ea men s. h ps://doi.o g/10.1038/s41467-020-17901-2 OPEN 1Cen e o Neu oscience and Cell Biology, Uni e si y o Coimb a, Coimb a, Po ugal. 2Facul y o Medicine, Uni e si y o Coimb a, Coimb a, Po ugal. 3Neu o ascula Resea ch Labo a o y, Vall d’Heb on Resea ch Ins i u e, Uni e si a Au ònoma de Ba celona, Passeig Vall d’Heb on 119-129, 08035 Ba celona, Spain. 4Aix Ma seille Uni , INSERM, MMG, Ma seille, F ance. 5In eg a i e Genomics o Ageing G oup, Ins i u e o Ageing and Ch onic Disease, Uni e si y o Li e pool, Li e pool L7 8TX, UK. 6P ogeli e, Ma seille, F ance. 7CECS, I-STEM, AFM, Ins i u e o S em Cell The apy and Explo a ion o Monogenic Diseases, E y Cedex, F ance. 8IMM, Ins i u o de Medicina Molecula , Uni e sidade de Lisboa, Lisbon, Po ugal. 925 Camb idge Science Pa k, Mog i y L d, Mil on Road, Camb idge CB4 0FW, UK. 10 Molecula Gene ics Labo a o y, Depa men o Medical Gene ics, La Timone Child en’s Hospi al, Ma seille, F ance. 11 De elopmen al Biology Labo a o y, F ancis C ick Ins i u e, London NW1 1AT, UK. 12 Leibniz Ins i u e on Aging - F i z Lipmann Ins i u e, 07745 Jena, Ge many. 13 CRB Assis ance Publique des Hôpi aux de Ma seille (CRB AP-HM, TAC), Ma seille, F ance. ✉email: [email p o ec ed] NATURE COMMUNICATIONS | (2020) 11:4110 | h ps://doi .o g/10.1038/s41467-020-17901-2 | www.na u e.com/na u ecommunica ions 1 1234567890():,; Hu chinson–Gil o d P oge ia Synd ome (HGPS) is caused by a single mu a ion in he lamin A/C gene (LMNA), esul ing in he gene a ion o an abno mal lamin A p e- cu so named p oge in1,2. One o he key easons o p ema u e dea h is he loss o smoo h muscle cells (SMCs) in he medial laye o la ge a e ies, ollowed by he appea ance o collagen and ex acellula ma ix (ECM) and he de elopmen o a se e e a e ioscle o ic p ocess ha leads o inc eased a e ial s i ness3–5. The easons o SMC loss emain o be de e mined. I has been sugges ed ha his may happen due o pa hophysiological changes inhe en o p elamin A/p oge in accumula ion, such as he accele a ion o ascula calcifica ion ia he ac i a ion o he DNA damage esponse and senescence-associa ed sec e o y pheno ypes in ascula SMCs6o he down egula ion o PARP17. I has also been shown ha he combined e ec o p oge in accumula ion and mechanical s ess in mouse SMCs o e - exp essing p oge in p omo ed cell de achmen and dea h, while he dis up ion o he linke be ween nucleoskele on and cy os- kele on complex amelio a ed he oxic e ec s o p oge in8. Nei- he o hese s udies ha e ully add essed he easons behind SMC de achmen and hus which he apeu ic app oach could be e ec i e o p e en SMC loss. Induced plu ipo en s em cells (iPSCs) o e an unlimi ed sou ce o SMCs o s udy HGPS. Recen s udies ha e gene a ed iPSCs om fib oblas s ob ained om indi iduals wi h HGPS (he ea e e e ed o as HGPS-iPSCs)9–11. S ikingly, HGPS- iPSCs show low lamin A/C and p oge in p o ein exp ession in he plu ipo en s a e. Howe e , he exp ession o p oge in is eac i- a ed a e HGPS-iPSC di e en ia ion in o SMCs7,9. The di e - en ia ed cells show nuclea dysmo phology, cell g ow h e a da ion, suscep ibili y o apop osis, p oli e a ion educ ion, and DNA- epai de ec s; howe e , SMC pe o mance unde flow condi ions has no been e alua ed. In his wo k, we de elop an in i o cell sys em comp ising SMCs de i ed om HGPS-iPSCs cul u ed unde flow condi ions in a mic ofluidic de ice. We iden i y MMP13 as a media o o SMC de achmen using chemical and gene ic assays. The gene - a ed double-mu an LmnaG609G/G609GMmp13−/−mice show an inc ease in SMCs in he ao ic a ch and a dec ease in p oge in- posi i e cells. In addi ion, he inhibi ion o MMP13 in LmnaG609G/G609G mice by Ba imas a , a d ug ha has been p e- iously es ed in clinical ials in cance pa ien s, educes SMC loss. The esul s p esen he e open pe spec i es o HGPS ea men . Resul s SMCs de i ed om HGPS-iPSCs a e unc ional and sha e simila ea u es o p oge in-exp essing cells. iPSCs we e gen- e a ed om HGPS skin fib oblas s and cha ac e ized as p e- iously desc ibed10. iPSCs gene a ed om non-disease cells (N- iPSCs), HGPS skin fib oblas s, and non-disease soma ic human ascula smoo h muscle cells (hVSMCs) we e used as con ols. The mu a ion in he LMNA gene, bo h in HGPS skin fib oblas s and HGPS-iPSCs, was confi med by Sange sequencing (Sup- plemen a y Fig. 1). As expec ed, undi e en ia ed HGPS-iPSCs exp essed low le els o HGPS ma ke s, such as p oge in, as well as low le els o SMC ma ke s, such as α-SMA and SMα-2212,13 (Supplemen a y Fig. 2a). To induce he di e en ia ion o HGPS- iPSCs o N-iPSCs in o SMCs, CD34+cells we e isola ed by magne ic-ac i a ed cell so ing om emb yoid bodies (EBs) cul- u ed o 10 days in suspension (Fig. 1a)14. A his s age, HGPS- CD34+cells al eady exp ess highe le els o p oge in mRNA ansc ip s ela i e o N-iPSCs bu ela i ely low le els o SMC mRNA ansc ip s compa ed wi h soma ic hVSMCs (Supple- men a y Fig. 2b). HGPS-CD34+cells we e hen cul u ed in SMC induc ion media (Supplemen a y Fig. 3) ollowed by SMC ma u a ion media (Supplemen a y Fig. 4) o an addi ional ou passages. Ma u ed SMCs a e e e ed o as HGPS-iPSC SMCs o N-iPSC SMCs based on hei pheno ype, geno ype, and unc- ional p ope ies (see below). Bo h HGPS-iPSC SMCs and N- iPSC SMCs ha e simila o highe exp ession o SMC mRNA ansc ip s han soma ic hVSMCs (Supplemen a y Fig. 4a). G ea e han 95% o bo h di e en ia ed cells exp ess α-SMA, smoo h muscle myosin hea y chain (SMMHC), and calponin p o eins (Fig. 1b). Mo eo e , HGPS-iPSC SMCs exp ess p oge in mRNA ansc ip s (Fig. 1c) and p oge in p o ein (Supplemen a y Fig. 4b, c). Simila esul s we e ob ained o SMCs de i ed om HGPS-iPSCs gene a ed om a second P oge ia indi idual; how- e e , he di e en ia ed cells showed highe p oge in p o ein le els han he fi s P oge ia indi idual (Supplemen a y Fig. 5). Impo an ly, HGPS-iPSC SMCs and N-iPSC SMCs a e unc ional as hey espond o asoac i e agen s such as his amine and angio ensin (Supplemen a y Fig. 4d) and hey con ac a e exposu e o ca bachol (Supplemen a y Fig. 4e). SMCs de i ed om HGPS-iPSCs sha e simila ea u es o p oge in-exp essing cells. Cell lines o ced o exp ess p oge in show he ac i a ion o se e al NOTCH signaling pa hway e ec o s15. Indeed, ou esul s showed ha HGPS-iPSC CD34+ cells had highe exp ession o NOTCH signaling pa hway mRNA ansc ip s han N-iPSC CD34+cells (Supplemen a y Fig. 6). Ma u e HGPS-iPSC SMCs also exp essed highe le els o NOTCH ligand and ecep o s han N-iPSC SMCs (Supplemen a y Fig. 6a). In addi ion, HGPS-iPSC SMCs esponded o a nesyl ans e ase inhibi o s, as has been shown in o he P oge ia cell models16–18. In he cu en wo k, HGPS-iPSC SMCs ea ed wi h lona a nib o 48 h accumula ed nuclea p elamin A and showed a dec ease in nuclea shape abno mali ies and nuclea blebbing (Supplemen a y Fig. 7a–c). Taken oge he , he cells di e en ia ed om HGPS- iPSCs-exp essed SMC and p oge oid ma ke s, a e unc ional and exhibi physiological esponses. HGPS-iPSC SMCs a e ulne able o a e ial shea s ess. SMCs di e en ia ed om N-iPSCs o HGPS-iPSCs we e seeded in a mic ofluidics sys em and cul u ed unde flow condi ions o up o 7 days (Fig. 1d). Because SMCs om la ge a e ies a e he mos a ec ed in blood essels in HGPS, we used a flow o 20 dyne/cm2, which is ypically ound in a e ial blood essels19. N-iPSC SMCs (Fig. 1g), hVSMCs, o HGPS fib oblas s (80% o which exp ess p oge in) (Fig. 1e, g) can be cul u ed in he mic ofluidics sys em o a leas 7 days wi hou a isible loss in cell numbe . In con- as , HGPS-iPSC SMCs cul u ed unde flow condi ions o med cell clumps o e ime (Fig. 1 ), and mos o he cells de ached om he subs a e a day 4 as confi med by cell numbe (Fig. 1g) and me abolic analyses (Fig. 1h). Du ing his ime pe iod, he pe - cen age o cells exp essing p oge in and displaying nuclea abno mali ies inc eased significan ly un il day 4 (Supplemen a y Fig. 8). Ou esul s indica e ha SMC de achmen is media ed by p oge in accumula ion, as he inhibi ion o p oge in by an isense mo pholinos20 significan ly dec eased HGPS-iPSC SMC de ach- men (Supplemen a y Fig. 9). In addi ion, we showed ha HGPS- iPSC SMCs wi h high p oge in exp ession (30% o he cells exp ess p oge in a day 0) de ached om he su ace o he mic ofluidics sys em in a sho ime (<12 h) (Supplemen a y Fig. 5g). To confi m ha p oge in accumula ion is esponsible o SMC loss, a ameshi mu an s em cell line was gene a ed (HGPSΔ2-iPSCs) o knockou he HGPS mu an allele and gen- e a ed a disease cell line, as p e iously desc ibed in he mouse21 (Fig. 2a and Supplemen a y Fig. 10). Specifically, a wo-base pai dele ion on exon 11, ups eam o he HGPS poin mu a ion (1814C>T), was gene a ed. No ably, HGPSΔ2-iPSCs exp essed ARTICLE NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-020-17901-2 2NATURE COMMUNICATIONS | (2020) 11:4110 | h ps://doi.o g/10.1038/s41467-020-17901-2 | www.na u e.com/na u ecommunica ions li le o no p oge in upon di e en ia ion in o SMCs as demon- s a ed a he ansc ip and p o ein le els and did no de ach unde flow cul u e condi ions (Fig. 2). HGPS-iPSC SMC de achmen does no seem o be media ed by cell apop osis. Be o e cell de achmen , HGPS-iPSC SMCs showed: (i) poo p oli e a ion (as moni o ed by Ki67 s aining) confi ming hei con ac ile pheno ype (Fig. 1i), (ii) simila le els o apop osis as N-iPSC SMCs as confi med by caspase-9 ac i i y (Fig. 1j), (iii) an os eogenic di e en ia ion p og am (Supplemen a y Fig. 11a, b), (i ) inc eased DNA damage6(Supplemen a y Fig. 12), and ( ) down egula ion o NOTCH15,22 (Supplemen a y Fig. 13) signal- ing pa hways. Because he in i o shea s ess om blood flow is no di ec ly sensed by SMCs bu by endo helial cells (ECs), we co- cul u ed SMCs di e en ia ed om HGPS-iPSCs (di ec ly a ached o he mic ofluidics subs a e) wi h human umbilical a e y ECs (HUAECs, on op o he SMCs) unde flow condi ions. Ini ially, we sc eened di e en cul u e condi ions and we ound ha endo helial g ow h media-2 (EGM-2) medium was a sui able medium o suppo bo h cells (Supplemen a y Fig. 14). Then, we co-cul u ed HUAECs and HGPS-iPSC SMCs a di e en a ios (1.6, 1, and 0.6) unde flow condi ions. In all he a ios es ed, we had a monolaye o HUAECs (Supplemen a y Fig. 15a) and HGPS-iPSC SMCs a ime ze o. A e 6 days in flow condi ions, a significan pe cen age (>40%) o HGPS-iPSC SMCs was los (Supplemen a y Fig. 15b). Fo he highes a io es ed (1.6), he loss o HGPS-iPSC SMCs occu ed wi hou isible loss o ECs. Ye , o EC:SMC a ios below 1, pa o ECs also de ached om he mic ofluidic chambe indica ing ha , a low ECs densi y, may u n ECs ulne able o flow condi ions. Impo an ly, cell ulne abili y o flow condi ions was only obse ed in co-cul u es *** ** 101 100 10–1 10–2 10–3 10–4 10–5 Rela i e gene exp ession LMNA LMNB1 P oge in N-iPSC-SMCs HGPS-iPSC-SMCs HGPS ib oblas s * **** **** **** 246 0 10,000 20,000 30,000 40,000 0 5 10 15 Cell clumps A ea o cell clumps Time (days) A ea o cell clumps (μm 2 ) Cell clumps (numbe /μm 2 )(x10 6 ) 04 04 04 0 50 100 No malized caspase 9 ac ici y HGPS-iPSC SMCs N-iPSC SMCs HGPS ib oblas s Time (days) **** * ** p < 0.0001 p = 0.0123 p = 0.0084 0246 0.0 0.5 1.0 1.5 2.0 Time in low shea s ess (days) No malized cell numbe HGPS-iPSCs-SMCs HGPS ib oblas s hVSMCs N-iPSCs-SMCs *** 0246 0.0 0.2 0.4 0.6 0246 Cell me abolism Cell p oli e a ion Cell apop osis Time in low shea s ess (days) Me abolic ac i i y hVSMCs HGPS-iPSCs-SMCs N-iPSCs-SMCs HGPS ib oblas s *** e h Day 1 Day 4 Day 6 Day 1 HGPS Fib oblas s Day 7 HGPS-iPSC SMCs hVSMCs Day 8 g 0 20 40 60 80 % Ki67 posi i e cells N-iPSCs-SMCs HGPS-iPSCs-SMCs HGPS ib oblas s 04 04 40 Time (days) **** **** * p < 0.0001 p < 0.0001 p = 0.0305 ij d a Days Cell coun ing 0 20 40 60 80 100 120 % o cells N-iPSC-SMCs HGPS-iPSC-SMCs α-SMA SMMHC Calp b Di e en ia ion in suspension CD34+ cells Undi e en ia ed cell colony 4 Passages (≈15 d) Induc i e media 4 Passages (≈15 d) Ma u a ion media HGPS-iPSC SMCs N-iPSC SMCs c NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-020-17901-2 ARTICLE NATURE COMMUNICATIONS | (2020) 11:4110 | h ps://doi .o g/10.1038/s41467-020-17901-2 | www.na u e.com/na u ecommunica ions 3 o HGPS-iPSC SMCs bu no N-iPSC SMCs (Supplemen a y Fig. 15c). I has been shown ha a knock-in mouse line ca ying a homozygous Lmna c.1827C>T;p.Gly609Gly mu a ed allele (LmnaG609G/G609G) ecapi ula es mos o he desc ibed al e a ions associa ed wi h HGPS, including he loss o SMCs20. Thus, o alida e he esul s ob ained o he HGPS-iPSC SMCs, we isola ed SMCs om wild- ype (WT mSMC) and homozygous LmnaG609G/G609G (HOZ mSMC) mice. Bo h cells exp essed calponin and α-SMA, while HOZ mSMCs, bu no WT SMCs, showed dysmo phic nuclei and nuclea blebbing (Fig. 3a, b). WT mSMCs we e cul u ed unde flow condi ions (120 dyne/cm2 o mimic mice a e ial flow shea s ess23,24) o up o 26 days wi hou isible loss o cells (Fig. 3c). In con as , HOZ mSMCs de ached om he subs a e a e 8–9 days. These esul s confi m ha HOZ mSMCs a e ulne able o flow shea s ess simila o HGPS-iPSC SMCs. O e all, ou esul s indica e ha HGPS-iPSC SMCs a e ulne able o flow shea s ess, as in he case o SMCs isola ed om mice ca ying a HGPS-like mu a ion in he Lmna gene. HGPS-iPSC SMCs ha e significan changes in ex acellula ma ix (ECM) sec e ion and MMP exp ession. To gain insigh s in o he mechanism behind SMC de achmen , we pe o med mic oa ay analyses on HGPS-iPSC SMCs and N-iPSC SMCs a days 0 and 4 (be o e cell de achmen ). A day 0, 2084 genes we e di e en ially exp essed (Log2FC ≥1; p< 0.05) in HPGS-iPSC SMCs s. N-iPSC SMCs. O hese genes, 51 genes we e associa ed wi h cell senescence, as de e mined by he in e sec ion o all he di e en ially exp essed genes wi h he CellAge da abase25 (279 genes) (Supplemen a y Da a 1). A he p o ein le els, HGPS-iPSC SMCs exp essed highe le els o p21 and SA-β-galac osidase han N-iPSCs-SMCs and he le el o senescence ma ke s inc eased a e cul u e o HGPS-iPSC SMCs in flow condi ions (Supple- men a y Fig. 16a and Supplemen a y Da a 5). We nex pe o med pa hway analysis on he di e en ially exp essed genes om HGPS-iPSC SMCs a day 0 s. day 4 (Supplemen a y Fig. 17 and Supplemen a y Da a 2, 3). In gene al, ECM ac i a ion, sec e ion, and cell adhesion pa hways we e up egula ed, whe eas cell cycle and DNA eplica ion pa hways we e down egula ed unde a e ial flow condi ions a day 4. Among he fi y-se en genes ha we e a leas h ee old down- o up egula ed compa ed wi h day 0 (p< 0.001) (Fig. 4a), fi e we e ela ed o ECM sec e ion (COL6A3, IBSP, BGN, SGCG, and EPPK1) and one o me allop o eases (MMP13). The exp ession o hese genes, as well as o he s, was confi med by qRT-PCR (Fig. 4a), and he molecula ne wo k o genes ha we e di e en ially exp essed be ween days 0 and 4 in he HGPS-SMCs was examined by Ingenui y Pa hway Analysis (Supplemen a y Fig. 17). In e es ingly, pa hway analysis sug- ges ed ha MMP13 is ei he a di ec o indi ec a ge o mul iple genes up egula ed a day 4. Mo eo e , MMP13 ansc ip le els a e ele a ed in HGPS-iPSC SMCs when compa ed wi h SMCs gene a ed om he a enua ed disease e sion o his line (HGPSΔ2-iPSC SMCs), specially pos shea s ess (Fig. 2g). To u he explo e he gene a ay esul s, we e alua ed whe he he p esence o ECM sec e ed by hVSMCs could p e en he de achmen o HGPS-iPSC SMCs unde a e ial flow condi ions. Thus, we cul u ed HGPS-iPSC SMCs on decellula ized ECM deposi ed by hVSMCs o di ec ly on op o mi o ically inac i a ed hVSMCs (Supplemen a y Fig. 18). Bo h condi ions we e unable o p e en HGPS-iPSC SMC de achmen . Nex , we es ed whe he condi ioned media collec ed om HGPS-iPSC SMCs in flow condi ions o 4 days could induce he de achmen o flow shea s ess-insensi i e hVSMCs (Fig. 4b). Su p isingly, hVSMCs de ach a e pe usion wi h HGPS-iPSC SMC-condi ioned media bu no wi h N-iPSC SMC-condi ioned media (Fig. 4c). Following hese esul s and gi en ha MMP13 appea s o be he downs eam e ec o o he genes mis egula ed a day 4 (Supplemen a y Fig. 17b) we decided o quan i y he concen a- ion o MMP13 in HGPS-iPSC SMC and N-iPSC SMC cul u e media a e flow shea s ess. Rema kably, MMP13 le els inc eased 30- old in he HGPS-iPSC SMC cul u e media, bu no in he con ol cell cul u e media (Fig. 4d). Simila ly, highe MMP13 le els we e obse ed in media collec ed om HOZ mSMCs unde flow shea s ess, when compa ed wi h media om WT mSMCs (Fig. 3d). Because MMP13 is p oduced by cells as an inac i e o m (p oMMP13), which is hen ac i a ed by cell memb ane MMPs, namely MMP14 (also called MT1-MMP) and MMP2 (also called gela inase A)26, he ca aly ic ac i i y o MMP13 sec e ed by HGPS-iPSC SMCs was analyzed (Supple- men a y Fig. 19). The concen a ion o p oMMP13 and ac i e MMP13 inc eased app oxima ely eigh - and fi e- old, espec- i ely, in cul u e media o HGPS-iPSC SMCs cul u ed in flow condi ions om day 0 o day 4. Mo eo e , he concen a ion o p oMMP13 and ac i e MMP13 in cell cul u e media collec ed om N-iPSC SMCs cul u ed in flow condi ions o 4 days was mo e han ou old lowe han he one obse ed wi h HGPS-iPSC SMCs. Al oge he , ou esul s indica e ha HGPS-iPSC SMCs cul u ed unde flow condi ions showed inc eased cell senescence, ECM ac i a ion, sec e ion, and cell adhesion pa hways up egula- ion and dys egula ion in he exp ession o MMP13. Fig. 1 Vulne abili y o HGPS-iPSC SMCs o a e ial flow condi ions. a Schema ic ep esen a ion o he me hodology used o di e en ia e iPSCs in o SMCs. bExp ession o SMC ma ke s on iPSC-de i ed SMCs. Pe cen age o posi i e cells exp essing SMC ma ke s as e alua ed by immunofluo escence (a leas 100 cells we e coun ed pe each ma ke ). Resul s a e mean ± SEM (n=3 independen expe imen s). cExp ession o p oge ia ma ke s on iPSC- de i ed SMCs. Gene exp ession by qRT-PCR (gene exp ession was no malized by he housekeeping gene GAPDH). HGPS fib oblas s we e used as con ol. Resul s a e mean ± SEM (n=4 echnical eplica es om a pool o h ee independen expe imen s). *, **, ***, **** deno e s a is ical significance (p< 0.05, p< 0.01, p< 0.001, p< 0.0001). S a is ical analyses we e pe o med by one-way ANOVA ollowed by Newman–Keuls’s pos es . dSchema ic ep esen a ion o he p o ocol used. Cells we e cul u ed o 6–8 days in a e ial flow condi ions (20 dyne/cm2). eLigh mic oscopy images o HGPS fib oblas s, hVSMCs, o HGPS-iPSC SMCs (10% o he cells accumula e p oge in p o ein) a di e en cul u e days. Only HGPS-iPSC SMCs de ached om he mic ofluidic sys em a day 4. Scale ba is 50 μm. Numbe and a ea o cell clumps in HGPS-iPSC SMCs a di e en imes (a leas wo images (×10) ha e been quan ified pe ime). Fo a ea o cell clumps n> 2 images examined o e h ee independen expe imen s; o cell clumps, n=3 independen expe imen s. gNumbe o cells pe su ace a ea (mm2) du ing cell cul u e unde a e ial flow (a leas h ee images (×10) ha e been quan ified pe ime; n=3–7 independen expe imen s). Cell numbe was no malized by he numbe o cells p esen a day 0. hCell me abolism e alua ed by he P es o Blue assay. Abso bance a 570 nm was measu ed and no malized o he 600-nm alues o he expe imen al wells. n=3 independen expe imen s. iExp ession o nuclea p oli e a ion ma ke , Ki67 (a leas h ee images (×10) ha e been quan ified pe ime). The pe cen age o Ki67 posi i e cells was e alua ed by immunofluo escence. n> 3 images examined o e h ee independen expe imen s. jCell apop osis e alua ed by caspase-9 ac i i y. Resul s we e no malized by cell numbe . n=3 independen expe imen s. F om c o g, esul s a e mean ± SEM. *, **, ***, **** deno e s a is ical significance (p< 0.05, p< 0.01, p< 0.001, p< 0.0001). S a is ical analyses we e pe o med by a wo- ailed unpai ed S uden ’s es iand j. ARTICLE NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-020-17901-2 4NATURE COMMUNICATIONS | (2020) 11:4110 | h ps://doi.o g/10.1038/s41467-020-17901-2 | www.na u e.com/na u ecommunica ions MMP13 media es HGPS-iPSC SMC loss unde flow condi ions. Nex , we es ed whe he he chemical inhibi ion o MMPs could p e en HGPS-iPSC SMC de achmen . Fo his pu pose, we used Ba imas a (BB-94)27, a b oad spec um ma ix me allop o ease inhibi o (IC50 =33 nM o MMP1328), and a specific MMP13 inhibi o py imidine-4,6-dica boxylic acid, bis-(4-fluo o-3- me hyl-benzylamide) (IC50 =8 nM)29. Rema kably, bo h inhi- bi o s significan ly dec eased he de achmen o HGPS-iPSC SMCs cul u ed unde a e ial flow condi ions (a leas un il day 12) (Fig. 4e), and his e ec was much supe io o ha o lona- a nib (Supplemen a y Fig. 7d) o inhibi ion h ough he py - ophospha e calcifica ion p ocess30 (Supplemen a y Fig. 11c). To confi m hese esul s, HGPS-iPSC SMCs we e subjec ed o siRNA knockdown o MMP13 and cul u ed unde a e ial flow condi- ions o 10 days (Fig. 4 , g). Ou esul s show ha he knock- down o MMP13 in SMCs inc eased he s abili y o HGPS-iPSC SMCs in flow cul u e condi ions compa ed wi h non- ea ed cells. We also analyzed he e ec s o MMP13 and BB94 inhibi ion in HOZ mSMCs (Fig. 3e). Simila o wha was obse ed wi h HGPS-iPSC SMCs, he de achmen was significan ly delayed when one o he inhibi o s was used. To u he demons a e he impo ance o MMP13 in HGPS-iPSC SMC de achmen , we en o ced he exp ession o MMP13 in soma ic SMCs (hVSMCs) and cul u ed he modified cells in flow cul u e condi ions 0246 0.0 0.5 1.0 1.5 2.0 No malized cell numbe (by day 0) Time (days) HGPS-iPSC SMCs HGPSΔ2-iPSC SMCs **** Calp SMMHC 0 30 60 90 120 % o cells Lamin A P oge in D.Nuclei Blebbing 0 30 60 90 120 % o cells LMNA G608G exp ession **** HGPS ib oblas s HGPS-iPSC SMCs HGPSΔ2-iPSC SMCs p < 0.0001 Mo phology Nuclei Lamin A Nuclei P oge in bNuclei SMMHC Nuclei Calponin cd e a 100 10–1 10–2 10–3 100 10–1 10–2 10–3 10–4 10–4 MMP13 exp ession HGPS-iPSC SMCs LMNA gene locus Exon 11 HGPS allele HGPS Poin Mu a ion Si e Ch omosome 1 sgRNA PAM 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ WT allele N-iPSCHGPS-iPSC HGPSΔ2-iPSC WT allele WT allele WT allele HGPS allele HGPSΔ2-iPSC SMCs 04 Time (days) ** ** *** p = 0.0035 p = 0.0002 p = 0.0016 g NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-020-17901-2 ARTICLE NATURE COMMUNICATIONS | (2020) 11:4110 | h ps://doi .o g/10.1038/s41467-020-17901-2 | www.na u e.com/na u ecommunica ions 5 Fig. 2 Exp ession o p oge ia and SMC ma ke s in HGPSΔ2-iPSC SMCs. a gRNA di ec s Cas9 nuclease agains mu a ed exon 11 o LMNA gene, ups eam he HGPS mu a ion, dis up ing p oge in, wi hou al e ing lamin A and lamin C. Sange sequencing o LMNA (NM_170707.4 ansc ip ) exon 11 was pe o med o : N-iPSCs, HGPS-iPSCs and HGPSΔ2-iPSCs, confi ming he dele ion o wo-base pai s in he HGPSΔ2-iPSCs. bExp ession o lamin A, p oge in, and SMC p o eins moni o ed by immunofluo escence. Scale ba is 100 μm. n=6 independen expe imen s. cExp ession o p oge in (LMNA G608G gene) in HGPS and HGPSΔ2 cell lines. Resul s a e mean ± SEM (n=4 echnical eplica es om a pool o h ee independen expe imen s). S a is ical analyses we e pe o med by a wo- ailed unpai ed S uden ’s es . dQuan ifica ion o lamin A, p oge in, dysmo phic nuclei, and nuclei blebbing. Resul s a e mean ± SEM (n=6 independen expe imen s). **** deno es s a is ical significance (p< 0.0001). ePe cen age o cells ha ha e been di e en ia ed om HGPSΔ2-iPSCs ha exp ess SMC ma ke s a p o ein le el. Resul s a e mean ± SEM (n=5–6 independen expe imen s). Numbe o cells pe su ace (mm2) as quan ified by high-con en mic oscopy (a leas h ee images (×10) ha e been quan ified pe ime). The numbe o cells was e alua ed a e 6 days unde a e ial flow and was no malized by he numbe o cells p esen a day 0. n> 3 images examined o e h ee independen expe imen s. gMMP13 mRNA ansc ip s quan ified by qRT-PCR analyses in HGPS-iPSC SMCs o HGPSΔ2-iPSC SMCs cul u ed unde flow condi ions. MMP13 mRNA ansc ip s we e no malized by GAPDH.n=4 echnical eplica es om a pool o h ee independen expe imen s. **, *** deno e s a is ical significance (p< 0.01, p< 0.001). S a is ical analyses we e pe o med by a wo- ailed unpai ed S uden ’s es . 08 08 0.00 0.01 0.02 0.03 Concen a ion o MMP13 (pg/cell) ** HOZ mSMCs WT mSMCs Time (days) p = 0.0022 D. Nuclei Blebbing Calponin α-SMA 0 20 40 60 80 100 0 20 40 60 80 100 % o cells % o cells wi h o ganized ibe s ab WT mSMCs HOZ mSMCs 0 5 10 15 0 20 40 60 80 100 120 Time (days) % o adhe ed cells HOZ HOZ MMP13i HOZ BB94 d Lamin A α-SMA 0 20 40 60 80 100 120 % o adhe ed cells Time (days) 03926 HOZ mSMC WT mSMC **** ce Fig. 3 Cha ac e iza ion and impac o flow shea s ess in SMCs isola ed om wild- ype (WT) and homozygous (HOZ) LmnaG609G/G609G mice. a Mouse SMCs we e cul u ed o 9–26 days in a e ial flow condi ions (120 dyne/cm2). Immunofluo escence analyses pe o med on mouse SMCs (6-week- old WT and HOZ Lmna G609G/G609G mice) a passage 4 o α-SMA and Lamin A. Nuclei we e s ained wi h DAPI. Scale ba is 20 µm. n=3–4 images examined o e h ee independen expe imen s. bPe cen age o dysmo phic nuclei, nuclei blebbing, and SMC o ganized fibe s in mSMCs (assessed in s a ic condi ions). n=3-4 images examined o e h ee independen expe imen s. cPe cen age o adhe ed cells o e ime. Cells we e cul u ed unde flow condi ions. n=3–4 independen expe imen s. S a is ical analyses we e pe o med by one-way ANOVA ollowed by Newman–Keuls’s pos es . dQuan ifica ion o MMP13 in HOZ mSMCs and WT mSMCs. Cells we e analyzed a day 0 and day 8 unde flow. Fluo escence signal was no malized by cell numbe . n=3–4 independen expe imen s. S a is ical analyses we e pe o med by a wo- ailed unpai ed S uden ’s es . ePe cen age o adhe ed cells o e ime. Cells we e cul u ed unde flow condi ions. n=5–6 independen expe imen s. In g aphs b–e, esul s a e mean ± SEM. *,**,***,**** deno e s a is ical significance (p< 0.05, p< 0.01, p< 0.001, p< 0.0001). ARTICLE NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-020-17901-2 6NATURE COMMUNICATIONS | (2020) 11:4110 | h ps://doi.o g/10.1038/s41467-020-17901-2 | www.na u e.com/na u ecommunica ions (Supplemen a y Fig. 19). No ably, he numbe o cells obse ed a day 7 is lowe han he one obse ed in WT cells indica ing ha some o he modified cells we e los du ing he flow cul u e condi ions. We hen asked whe he he modula ion o MMP13 ac i i y could a ec p oge in exp ession associa ed wi h he ulne abili y o HGPS-iPSC SMCs o flow shea s ess. In e es ingly, chemical inhibi ion o MMP13 in HGPS-iPSC SMCs cul u ed o 7 days in flow condi ions educed he pe cen age o p oge in-posi i e cells (Fig. 4h); howe e , i did no dec ease p oge in exp ession in cells wi h high le els o p oge in, such as HGPS fib oblas s. In addi ion, he chemical inhibi ion o MMP13 did no educe he ac i i y o alkaline phospha ase in HGPS-iPSC SMCs cul u ed o 7 days in flow condi ions (Fig. 4i). O e all, he esul s ob ained 012 24 36 48 0 25 50 75 100 125 150 Time (h) % o adhe ed hVSMCs HGPS-iPSC SMC CM N-iPSC SMC CM Non- ea ed BB94 MMP13i BB94 MMP13i 0.0 04040404 0.5 1.0 1.5 2.0 No malized cell numbe o day 0 d7 d12 **** **** **** **** 0 50 100 150 % o cells wi h p oge in HGPS ib oblas s HGPS-iPSC SMCs MMP13 inhibi ion –+– + **** p < 0.0001 0.00 0.01 0.02 0.03 0.04 Rela i e gene exp ession *** No ans ec ion siRNA con ol siRNA MMP13 *** 0 2 4 6 8 Concen a ion o MMP13 (x10–4, pg/cell) **** * * Time (days) HGPS-iPSC SMCs N-iPSC SMCs hVSMCs HGPS ib oblas s p < 0.0001 p = 0.0156 p = 0.0417 EPS8 NR4A3 PXK OLFML2A LIPH AMTN TNFAIP6 ANGPTL4 EPPK1 SESN2 SGCG MMP13 IBSP HAS3 COL6A3 BGN –4 –2 0 2 4 6 8 10 12 14 Fold change HGPS-iPSC SMCs N-iPSC SMCs *** **** *** ** *** **** ** * ** ** *** ** **** * **** p = 0.0023 –6–4–20246 10–7 10–6 10–5 10–4 10–3 10–2 10–1 100 Log2 ( old change) 4 days Concen a ion o MMP13 E ec o media in he de achmen o hVSMCs MMP13 inhibi ion 4 – 12 days Cell de achmen P oge in accumula ion Alkaline phospha ase ac i i y ec b d 710 0 10 20 30 40 Time (days) Fold o con ol ( ans ec ed wi h con ol siRNA) hi 0 1 2 3 4 5 Alkaline phospha ase ac i i y no malized by cell numbe MMP13 inhibi ion +– a p- alue g NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-020-17901-2 ARTICLE NATURE COMMUNICATIONS | (2020) 11:4110 | h ps://doi .o g/10.1038/s41467-020-17901-2 | www.na u e.com/na u ecommunica ions 7 a e chemical and gene ic inhibi ion, he inc ease o MMP13 a e flow shea s ess and he e ec o HGPS-iPSC SMC- condi ioned media on cell de achmen , indica e ha MMP13 media es SMC loss. Inhibi ion o MMP13 in LmnaG609G/G609G mice significan ly inc eased he numbe o SMCs in ao ic a ch.Toconfi m he impo ance o MMP13 dys egula ion in p oge oid animal models, we quan ified MMP13 in he plasma o LmnaG609G/G609G and WT mice (Fig. 5a). The esul s showed ha he le els o MMP13 we e highe in mu an mice (Fig. 5b). Then, we asked whe he he inhibi ion o MMP13 in LmnaG609G/G609G mice could dec ease SMC loss. Fo his pu pose, we gene a ed double-mu an lines, LmnaG609G/G609GMmp13−/−and LmnaG609G/G609GMmp13+/− (Supplemen a y Fig. 20), and e alua ed he hea a e and SMC loss in he ao ic a ch20 o hese mice a week 10 (Fig. 5a). Hea a e waschosenasameasu eo heo e allheal hs a uso heHGPS model and he de i ed double-mu an lines, gi en ha b adyca dia was a clinical abno mali y e idenced in bo h LmnaG609G/G609G mouseaswellasZmps e24 −/−p oge ia mouse models20,31. Bo h double-mu an mice showed highe hea a es (Fig. 5d) and numbe s o SMCs (Fig. 5c, e) in he ao ic a ch han LmnaG609G/G609G Mmp13+/+mice. In e es ingly, LmnaG609G/G609G Mmp13−/−and LmnaG609G/G609GMmp13+/−mice showed a lowe numbe o p oge in-posi i e cells in he ao ic a ch han non- mu a ed mice (Fig. 5c, ). In addi ion, LmnaG609G/G609GMmp13+/− mice (bu no LmnaG609G/G609GMmp13−/−mice) showed an inc ease o he ao ic media hickness being simila o he non- mu a ed mice (Fig. 6a), as confi med by o cein s aining. We pe o med p o eomic analyses o ao ic a ches om mu a ed and non-mu a ed mice (n≥5 mice pe s ain) using da a independen acquisi ion mass spec ome y32,33. P incipal componen analysis based on 2260 p o eins de ec ed showed ha he p o eome p ofiles o ao ic a ches om LmnaG609G/G609GMmp13+/−mice we e mo e closely ela ed o he p ofile o WT mice o ha o LmnaG609G/ G609G Mmp13+/+mice (Fig. 6c). F om he 161 p o eins di e en- ially exp essed be ween he mu an and WT mice ao ic a ches (q <0.05andabs(log 2 old change) > 0.58), ~25% o he p o eins had simila exp ession in LmnaG609G/G609GMmp13+/−mice and WT mice (Fig. 6c and Supplemen a y Da a 4). Mo i a ed by hese esul s, we hen es ed a he apeu ic app oach o educe SMC loss in LmnaG609G/G609G Mmp13+/+ mice. Fo his pu pose, we used Ba imas a because human sa e y has been p e iously demons a ed in clinical ials34. LmnaG609G/G609G Mmp13+/+mice a week 5 we e in ape i oneal (IP) injec ed fi e imes a week (Fig. 7a). A week 10, Ba imas a - ea ed LmnaG609G/G609G Mmp13+/+mice had simila hea a es o non- ea ed animals (Fig. 7c); howe e , hey showed highe SMCs in he ao ic a ch han non- ea ed mice, as confi med by cell nuclei coun s and e ified by he inc ease le els o SMC ma ke s de e mined by qRT-PCR analyses (Fig. 7b, d, e). No di e ences we e obse ed be ween non- ea ed and Ba imas a - ea ed mice ega ding p oge in accumula ion in he ao ic a ch (Supplemen a y Figs. 20c). O e all, ou da a shows ha he in i o inhibi ion o MMP13 by gene ic o chemical in e en ions yielded mice ha ing significan ly highe numbe s o SMCs in he ao ic a ch. Ac i a ion o MMP13 is media ed by he ac i a ion o he glycocalyx. The glycocalyx is a su ace laye o p o eoglycans and glycosaminoglycans ha a e immobilized in he cell memb ane. Glycocalyx componen s ha e been shown o be in ol ed in flow shea s ess sensing by SMCs35,36. To iden i y he mechanism unde lying he up egula ion o MMP13 in HGPS-iPSC SMCs cul u ed unde a e ial flow, we analyzed glycocalyx gene mRNA ansc ip s (Fig. 8b). In e es ingly, glycocalyx ansc ip s we e up egula ed in HGPS-iPSC SMCs cul u ed unde flow condi ions o 4 days (Fig. 8b). F om hese up egula ed genes, syndecan 2 gene (SDC2), which encodes he ansmemb ane ( ype I) hepa an sul a e p o eoglycan, was also up egula ed in hVSMCs o N-iPSC SMCs cul u ed o 4 days in flow condi ions (Supplemen a y Fig. 21). Because no all he glycocalyx mRNA ansc ip s we e up egula ed in hVSMCs and N-iPSC SMCs, he esul s sugges ha he composi ion o glycocalyx is likely di e en in hese cells when compa ed wi h HGPS-iPSC SMCs. Nex , we analyzed he exp ession o hepa an sul a e a he p o ein le el. In con as o con ol cells, he exp ession o hepa an sul a e inc eased when HGPS-iPSC SMCs we e cul u ed unde flow condi ions (Fig. 8a). Impo an ly, he enzyma ic clea age o hepa an sul a e by hepa inase III (Supplemen a y Fig. 22) dec eased MMP13 con- cen a ion in he cell cul u e media (Fig. 8c) and significan ly dec eased he de achmen o HGPS-iPSC SMCs cul u ed unde flow condi ions (Fig. 8d). Mo eo e , he enzyma ic clea age o hepa an sul a e sligh ly dec eased alkaline phospha ase ac i i y (Fig. 8e). To u he in es iga e a po en ial ECM a ge o MMP13 in SMCs, we moni o ed he exp ession o ECM componen s in hVSMCs, HUAECs, N-iPSC SMCs, and HGPS-iPSC SMCs. Ou Fig. 4 MMP13 ac i i y in HGPS-iPSC SMCs cul u ed unde flow shea s ess. a Volcano plo ep esen ing di e en ially exp essed genes in HGPS-iPSC- SMC cul u ed unde flow condi ions a day 0 and 4. Each poin ep esen s one o 53,617 genes. 26 and 31 genes we e up egula ed ( ed; old change ≥3; p< 0.001) and down egula ed (yellow; old change ≤3; p< 0.001), espec i ely. G aph shows qRT-PCR alida ion o 16 genes wi h old changes >3. Fold change was be ween days 0 and 4. Gene exp ession was no malized by he housekeeping gene GAPDH. Resul s a e mean ± SEM, n=4 echnical eplica es om a pool o h ee independen expe imen s. S a is ical analyses we e pe o med by a wo- ailed unpai ed S uden ’s es . bSchema ic ep esen a ion o he expe imen al p o ocol used. cE ec o HGPS-iPSC SMC o N-iPSC SMCs condi ioned media (in bo h cases ob ained a e 4 days unde flow condi ions) on hVSMCs cul u ed unde flow condi ions. n=1–5 images examined o e h ee independen expe imen s. dQuan ifica ion o MMP13 ac i i y (cell cul u e media) by ELISA. Cells we e analyzed a days 0 and 4 unde flow. Fluo escence signal was no malized by cell numbe . n=3 independen expe imen s. S a is ical analyses we e pe o med by a wo- ailed unpai ed S uden ’s es . eE ec o MMP13 o BB94 inhibi ion in HGPS-iPSC SMC de achmen . The numbe o cells was e alua ed a e 7 and 12 days unde a e ial flow and was no malized by he numbe o cells p esen a day 0. n=3–5 images examined o e h ee independen expe imen s. S a is ical analyses we e pe o med by one-way ANOVA ollowed by Newman–Keuls’s pos es . MMP13 knockdown by siRNA in HGPS-iPSC SMCs. MMP13 mRNA ansc ip s we e quan ified by qRT-PCR and no malized by GAPDH.Mean±SEM(n=4 echnical eplica es om a pool o h ee independen expe imen s). S a is ical analyses we e pe o med by one-way ANOVA ollowed by Newman–Keuls’s pos es . gNumbe o cells pe mic ofluidic a ea du ing cul u e unde flow shea condi ions no malized by he numbe o cells in con ol expe imen al g oups (i.e., cells ans ec ed wi h con ol siRNA). n=7 independen expe imen s o day 7 and n=6 independen expe imen s o day 10. hPe cen age o p oge in-posi i e cells a e 7 days unde flow condi ions wi h SmGM-2 media supplemen ed o no wi h MMP13 inhibi o . n=1–5 images examined o e h ee independen expe imen s. S a is ical analyses we e pe o med by a wo- ailed unpai ed S uden ’s es . iAc i i y o alkaline phospha ase in HGPS- iPSCs-SMC no malized by cell numbe pe mm2, in cells cul u ed 4 days unde flow condi ions. Cells we e ea ed o no wi h MMP13 inhibi o . n=3 independen expe imen s. In g aphs a–h, esul s a e mean ± SEM. *, **, ***, **** deno e s a is ical significance (p< 0.05, p< 0.01, p< 0.001, p< 0.0001). ARTICLE NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-020-17901-2 8NATURE COMMUNICATIONS | (2020) 11:4110 | h ps://doi.o g/10.1038/s41467-020-17901-2 | www.na u e.com/na u ecommunica ions 0 10 20 30 40 50 Concen a ion o MMP13 no malize by weigh *** KiW W W p = 0.0001 d W W KiW e a KiH α-SMA Nuclei c 555 P oge in NucleiHS Nuclei 0 200 400 600 Hea a e (bpm) W W KiW KiH KiKO ** * b 0.0 0.5 1.0 1.5 2.0 2.5 In ensi y HS (a.u.)/cell numbe (no malized by W W ) KiKO g 0.0 0.5 1.0 1.5 2.0 2.5 α-SMA-posi i e cell nuclei/mm2 (no malized by W W ) * * ** p = 0.003 p = 0.0343 p = 0.0142 0 2 4 6 8 10 % P oge in posi i e cells (no malized by W W ) **** * * ** ** Lmna+/+ = W W LmnaG609G/G609G Mmp13+/+ = KiW LmnaG609G/G609G Mmp13+/– = KiH LmnaG609G/G609G Mmp13–/– = KiKo Age: 10 weeks Hea a e Immunohis ochemis y Fig. 5 MMP13 inhibi ion significan ly inc eases SMC numbe in ao ic a ch o LmnaG609G/G609G mice. a Schema ic ep esen a ion o he animal p o ocol. W W , KiW , KiH , and KiKo mice (age: 10 weeks) we e e alua ed. bQuan ifica ion o MMP13 ac i i y (plasma om W W , n=9, and KiW , n=6, mice) by ELISA. Fluo escence signal was no malized by mice weigh . S a is ical analyses we e pe o med by a wo- ailed unpai ed S uden ’s es . c Immunofluo escence analyses in he ao ic a ch o α-SMA, p oge in, and hepa an sul a e (HS). Cell nuclei we e s ained wi h DAPI. Scale ba is 100 µm o α-SMA s aining and 50 µm o p oge in and hepa an sul a e s aining. Fo α-SMA s aining, n=5 animals, excep o KiH ( ou animals). Fo p oge in s aining, n=5 animals, excep o KiH ( h ee animals). Fo hepa an sul a e n=6 W W , n=6 KiW , n=4 KiH , and n=5 o KiKo. dHea a es in mice (n=8 W W , n=6 KiW , n=7 KiH , and n=5 KiKo). S a is ical analyses we e pe o med by one-way ANOVA ollowed by Newman–Keuls’s pos es . eNumbe o SMC nuclei in ao ic a ch pe issue a ea (mm2)(n=2–3 slides examined o e fi e animals, excep o KiH ( ou animals)). S a is ical analyses we e pe o med by a wo- ailed unpai ed S uden ’s es . Pe cen age o p oge in-posi i e cells in SMCs. n=5 animals, excep o KiH ( h ee animals). S a is ical analyses we e pe o med by one-way ANOVA ollowed by Newman–Keuls’s pos es . gExp ession o hepa an sul a e as e alua ed by immunofluo escence. In ensi y o hepa an sul a e was calcula ed in each pic u e (a leas 16 pic u es pe condi ion) and no malized by cell numbe mice (n=6 W W , n=6 KiW , n=4, KiH and n=5 KiKo). In b,d–g, esul s a e mean ± SEM. *, **, ***, **** deno e s a is ical significance (p< 0.05, p< 0.01, p< 0.001, p< 0.0001). NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-020-17901-2 ARTICLE NATURE COMMUNICATIONS | (2020) 11:4110 | h ps://doi .o g/10.1038/s41467-020-17901-2 | www.na u e.com/na u ecommunica ions 9 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 ea 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 . To iew a copy o his license, isi h p://c ea i ecommons.o g/ licenses/by/4.0/. © The Au ho (s) 2020 ARTICLE NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-020-17901-2 16 NATURE COMMUNICATIONS | (2020) 11:4110 | h ps://doi.o g/10.1038/s41467-020-17901-2 | www.na u e.com/na u ecommunica ions