ABSTRACT
MMP1 - 1 6 0 7 poly m or ph ism in cr eases t h e r isk
for per iapical lesion developm ent t hr ough t he
upr egulat ion MMP- 1 ex pr ession in associat ion
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Ana Paula Favaro TROMBONE1 )UDQFR &$9$//$2,3 (OFLD 0DULD 9DUL]H 6,/9(,5$1, Camile Bermejo ANDREO1, Carolina Favaro FRANCISCONI2, Angélica Cristina FONSECA2, Ariadne LETRA45HQDWR0HQH]HV6,/9$4, Gustavo Pompermaier GARLET2
1- Universidade do Sagrado Coração, Departamento de Ciências Biológicas e da Saúde, Bauru, SP, Brasil.
2- Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Ciências Biológicas, Bauru, SP, Brasil. 3- Universidad de Chile, Facultad de Odontología, Departamento de Odontología Conservadora, Santiago, Chile.
4- University of Texas Health Science Center at Houston, School of Dentistry, Department of Endodontics, Houston, USA.
Corresponding address: Gustavo Pompermaier Garlet - Faculdade de Odontologia de Bauru - Departamento de Ciências Biológicas - Al. Octávio Pinheiro Brisola, 9-75 - 17012-901 - Bauru - SP - Brazil - Phone +55 (14) 3235-8274 - Fax +55 (14) 3223-4679 - e-mail: [email protected]
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ncreased m at rix m et alloprot einases ( MMPs) act ivit y is a hallm ark of periapical granulom as. However, t he fact ors underlying t he MMPs expression m odulat ion in healt hy and diseased periapical t issues rem ains t o be det erm ined. Obj ect ive: I n t his st udy, w e evaluat ed t heDVVRFLDWLRQEHWZHHQWKH003SRO\PRUSKLVPUVDQGSURLQÀDPPDWRU\
m ilieu elem ent s w it h MMP- 1 m RNA levels in vivo. Mat erial and Met hods: MMP1- 1607 SNP and t he m RNA levels of MMP- 1, TNF-D, I FN-J, I L- 17A, I L- 21, I L- 10, I L- 4, I L- 9, and FOXp3 w ere det erm ined via RealTim ePCR in DNA/ RNA sam ples from pat ient s present ing periapical granulom as ( N= 111, for bot h genot yping and expression analysis) and cont rol subj ect s ( N= 214 for genot yping and N= 26 for expression analysis) . The Shapiro-Wilk, Fisher, Pearson, Chi- square ordinal least squares regression t est s w ere used for dat a
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cont rols, com prising a risk fact or for periapical lesions developm ent . MMP- 1 m RNA levels w ere higher in periapical lesions t han in healt hy periodont al ligam ent sam ples, as w ell as higher in act ive t han in inact ive lesions. The polym orphic allele 2G carriers present ed a
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genet ic polym orphism and t he expression levels of MMP- 1. Addit ionally, t he pro- and ant
i-LQÀDPPDWRU\F\WRNLQHV,/$,)1J, TNF-D,/,/,/DQG,/ZHUHVLJQL¿FDQW as com plem ent ary explanat ory variables of MMP- 1 expression. Conclusion: The MMP1- 1607
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associat ion wit h increased MMP- 1 m RNA levels in periapical lesions. The MMP- 1 expression
LVDOVRXQGHUWKHFRQWURORIWKHLQÀDPPDWRU\PLOLHXHOHPHQWVEHLQJWKHF\WRNLQHV71)D, I L- 21, I L- 17A, and I FN- g associat ed wit h increased MMP- 1 levels in periapical lesions, while I L- 10, I L- 9, or I L- 4 present ed an inverse associat ion.
Ke y w o r d s: Per iap ical d iseases. Per iap ical g r an u lom a. Mat r ix m et allop r ot ein ases.
I N TROD UCTI ON
Per iap ical lesion s ar e ch ar act er ized b y t h e dest ruct ion of periapical t issues as a consequence of t h e local h ost r esp on se, w h ich is t r ig g er ed by bact er ial infect ion of pulpal and en dodont ic environm ent18,30. Am ong m ult iple elem ent s involved
LQKRVWLQÀDPPDWRU\LPPXQHUHVSRQVHVFyt okines a r e cr i t i ca l d et er m i n a n t s o f l esi o n s o u t co m e act ing as m aj or m odulat ors of t he m ain pat hways r esponsible for t issue dest r uct ion13, 18. While t he RANK- RANKL- OPG sy st em , t he m ain r esponsible for ost eoclast ogenesis cont rol, is supposed t o be one det erm inant of periapical lesions act ivit y, t he balance bet ween MMPs ( m at rix m et alloprot einases, a fam ily of zinc- and calcium - dependent prot eases) and TI MPs ( t he endogenous t issue inhibit or s of m et allop r ot ein ases) is su p p osed t o d et er m in e t he out com e of t he pr ot ease- m ediat ed cat abolic r esponse t hat cont r ibut es t o t he degradat ion of
soft and m ineralized t issues surrounding t he root apex18,19,26,34,.
I n physiological circum st ances, MMPs play a role in degradat ion and rem odeling of bot h ext racellular and bone m at rix prot eins. How ever, t he increase in t he expression and/ or act ivat ion of MMPs w it hout a parallel increase of TI MPs t o count eract cat abolic p r o t e o l y s i s m e d i a t e n u m e r o u s p a t h o l o g i c a l processes, including t he periapical and periodont al diseases6,23,31. I ndeed, t he high m at rix prot einase act iv it y is a hallm ar k of per iapical granulom as, and is supposed t o derive from a com bined act ion of m ult iple MMPs such as MMP- 1, MMP- 2, MMP- 8, MMP- 9, and MMP- 131,28.
Wit hin t he MMPs found t o be overexpressed in
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con t r ibu t e t o lesion dev elopm en t by m ediat in g
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since MMP- 1 is t he m ain prot eolyt ic enzym e t hat
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t issue m at rix m ost abundant com ponent s. I ndeed, w hile MMP- 1 levels ar e r elat ively low in healt hy per iapical t issues, it s lev els ar e upr egulat ed in h u m an an d ex per im en t al per iapical lesion s1 2 , 2 0. I t is im port ant t o m ent ion t hat a high individual variat ion in t he levels of MMP- 1 was described in
periapical lesions297KHUHIRUHVLQFHPRGL¿FDWLRQV
i n MMP- 1 l e v e l s m a y d i r e ct l y i n f l u e n ce t h e
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of t he fact ors t hat det erm ine t he cont rol of MMP- 1 expression in periapical environm ent m ay com prise im p or t an t in f or m at ion t o p r ed ict an d p r ev en t lesions developm ent as w ell t o develop t herapeut ic st rat egies t o lim it lesions progression.
Var iat ion s in t h e p r om ot er r eg ion of MMP1 gene account for her it able differ ences in
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t h e su scept ibilit y t o cer t ain pat h ologies4 , 2 9 , 3 2. A
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- 1607 posit ion of MMP1 gene pr om ot er ( MMP1-1607, rs: 1799750) is part icularly associat ed w it h increased MMP- 1 m RNA t ranscript ional act ivit y29,32. W h i l e p r e v i o u s st u d i e s su g g e st t h a t g e n e t i c variat ions in MMP- 2 and MMP- 3 genes account for increased risk t o periapical lesion form at ion26, t he possible associat ion of MMP- 1 genet ic variant s was not invest igat ed.
,Q DGGLWLRQ WR WKH LQÀXHQFH RI KRVW JHQHWLF background, it is also im port ant t o consider t hat d i f f er en t cy t o k i n es m ay al so acco u n t f o r t h e var iance of MMP- 1 lev els in per iapical t issues2.
I n d eed , st r o n g a n d p er si st en t m i cr o b i a l a n d LQÀDPPDWRU\VWLPXOLZHUHGHVFULEHGWRRYHUULGHWKH genet ic cont rol of MMP- 1 expression in periodont it is cont ext29. Accordingly , SURLQÀDPPDWRU\F\WRNLQHV
are described t o direct ly increase t he levels of MMP-ZKLOHDQWLLQÀDPPDWRU\F\WRNLQHVDUHVXSSRVHG t o count eract such effect and t o downregulat e MMPs expression8,14. I nt erest ingly, a m arked dichot om y
EHWZHHQ SUR DQG DQWLLQÀDPPDWRU\ F\WRNLQHV clu st er s is associat ed w it h p er iap ical act iv it y / inact ivit y st at us3, suggest ing t hat such m ediat ors
could in fact det er m ine lesions act iv it y v ia t he regulat ion of MMPs expression3.
Therefore, in t he present st udy w e invest igat ed
t h e p o ssi b l e a sso ci a t i o n o f MMP1 - 1 6 0 7 SNP w it h p er iap ical lesion s d ev elop m en t r isk , an d
si m u l t a n e o u sl y a n a l y ze d t h e i m p a ct o f su ch polym orphism and host response m ediat ors ( TNF, I L- 21, I L17, I FN- g, I L- 10, FOXp3, I L- 9, and I L- 4) in t he m odulat ion of MMP- 1 m RNA t ranscript ion in hum an chronic periapical granulom as.
M ATERI AL AN D M ETH OD S
Su bj e ct s a n d sa m ple s
Th i s st u d y h a d i n st i t u t i o n a l r e v i e w b o a r d approval of Bauru School of Dent ist ry, Universit y o f Sã o Pa u l o . Pa t i e n t s a n d co n t r o l su b j e ct s w er e select ed as pr ev iously descr ibed3. Pat ient s p r esen t in g p er iap ical lesion s w er e r ef er r ed t o endodont ic surgery aft er convent ional root canal t r eat m en t f ailu r e; p er iap ical lesion s d iag n osis was perform ed as previously described25 based on hist opat hological and radiographic analysis, being periapical lesions charact erized radiographically as rarefact ion lesions w it h t he disappearance of t he periodont al ligam ent space and discont inuit y of t he
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Per iapical gr an u lom as ( N= 1 1 1 ) w er e collect ed f r om p at ien t s ( N= 1 1 1 , ag ed 1 9 - 5 9 y ear s; 5 1 fem ales and 59 m ales) during periapical surgery and divided in t w o roughly sim ilar fragm ent s and st or ed in bot h for m alin ( for r out ine hist ological ex am inat ion per for m ed aft er hem at ox y lin- eosin st aining) and RNAlat er solut ion ( Am bion - Therm o
)LVKHU 6FLHQWL¿F ,QF Walt ham , Massachuset t s, USA) ( for m olecular analysis) . Test sam ples w ere
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m acr oph ages, an d w it h ou t t h e pr esen ce of an epit helial lining. Per iapical cy st s, w her e cav it ies
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squ am ou s epit h eliu m , an d par t ially epit h elized lesions ( epit helized granulom as) w er e excluded f r o m t h e st u d y. Per i a p i ca l l esi o n s w er e a l so cat egorized int o put at ive act ive and inact ive, based in t h e m olecu lar p r of ile of RANKL/ OPG m RNA ex pr ession , as pr ev iou sly descr ibed2 4. Pat ien t s’ epit helial buccal cells w er e sam pled fr om inner cheek buccal m ucosa scrapping aft er a m out hwash w it h 3% glucose for genot yping purposes.
Th e con t r ol g r ou p ( N= 2 1 4 ) ( p at ien t s ag ed 18- 52 year s; 103 fem ales and 111 m ales) was com pr ised of subj ect s w it hout a clinical hist or y of pulpal or per iapical pat hology and w er e fr ee fr om per iapical lesions. Epit helial buccal cells of cont rol subj ect s w ere sam pled from inner cheek buccal m ucosa scrapping aft er a m out hwash w it h 3% glucose for genot yping purposes. A subgroup of cont rol subj ect s ( N= 26) ( pat ient s aged 18- 35 years; 13 fem ales and 11 m ales) was com prised of subj ect s scheduled for prem olar ext ract ion due t o ort hodont ic purposes; in addit ion t o epit helial buccal cells sam pling, during t he surgery periapical t issue sam ples w ere isolat ed, st ored in RNA lat er solut ion and used as cont rol specim ens.
Pat ient s and cont r ols w it h m edical condit ions
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m et abolism or ot her assist ed dr ug t herapy ( i.e. V\VWHPLFDQWLELRWLFVDQWLLQÀDPPDWRU\KRUPRQDO
t herapy ) dur ing t he last six m ont hs befor e t he st udy w ere excluded. Pat ient s and cont rols w it h preexist ing condit ions, such as periodont al disease an d p r eg n an t o r l act at i n g w o m en , w er e al so excluded.
D N A e x t r a ct ion a n d a n a lysis of M M P1 - 1 6 0 7 SN P ( r s1 7 9 9 7 5 0 )
D NA w a s ex t r a ct ed f r o m ep i t h el i a l b u cca l cells w it h sequent ial phenol/ chlor ofor m solut ion as p r ev iou sly d escr ib ed1 1. Ex t r act ed DNA w as used for genot yping. DNA int egrit y was checked and t he allelic discrim inat ion of MMP1- 1607 SNP
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I nc. ; Walt ham , Massachuset t s, USA) chem ist r y
as p r ev iou sly d escr ib ed2 1. For r eact ion q u alit y cont rol, a sam ple of know n genot ype was included in t he plat e and a no DNA t em plat e sam ple was included as negat ive cont rol. Only genot ypes w it h an aut om at ic call rat e > 95% were considered, error rat e was < 3% . Sam ples t hat failed t o provide a genot ype w er e r epeat ed in addit ional r eact ions; genot yping was perform ed blinded t o group st at us, as previously described7.
RN A e x t r a ct ion a n d Re a lTim e - PCR
I n brief, t ot al RNA was ext ract ed from sam ples by using t he RNeasy k it ( Qiagen I nc, Valencia, California, USA) according t o t he m anufact urers’ inst r uct ions. The int egr it y of RNA sam ples w as
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Bioanaly zer ( Agilent Technologies, Sant a Clara, California, USA) according t o t he m anufact urers’ inst ruct ions. Aft er RNA ext ract ion, com plem ent ary
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a reverse t ranscript ion react ion using Quant iTect RT k it ( Qiagen I n c, Valen cia, Calif or n ia, USA) . All cyt okines/ Th m arkers (TNF-D ,)1DŽI 17A, I L-21, I L- 10, I L- 4, I L- 9, FOXp3) m RNA levels w ere m easured by m eans of RealTim ePCR using TaqMan
FKHPLVWU\7KHUPR)LVKHU6FLHQWL¿F,QFWalt ham , Massachuset t s, USA) in a Viia7 inst rum ent ( Therm o
)LVKHU 6FLHQWL¿F ,QF Walt ham , Massachuset t s, USA) using invent oried opt im ized prim ers/ probes set s ( I nvit rogen, Carlsbad, CA) , wit h basic react ion condit ions ( 40 cycles) 95° C ( 10’) , 94° C ( 1’) , 56° C ( 1’) , and 72° C ( 2’) . The analysis of RANKL and OPG m RNA levels were also det erm ined in all t he lesions ( also by RealTim ePCR using TaqMan chem ist ry) , in order t o cat egorize each sam ple in put at ive act ive and inact ive lesions based on t he RANKL/ OPG rat io as previously described24. The result s are depict ed as t he relat ive level of gene expression; calculat ed
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expression in each sam ple using t he 2¨¨Ct m et hod.
D a t a a n a lysis
int errelat ion bet ween t he MMP1- 1607 genot ype and t he MMP- 1 m RNA levels was assayed by ordered logist ic regression. The relat ive cont ribut ion of t he explanat ory variables MMP1- 1607 genot ype and t he H[SUHVVLRQOHYHOVRIWKHSURLQÀDPPDWRU\71)D, ,/$ ,/ DQG ,)1 DQG DQWLLQÀDPPDWRU\ ( Fox P3 , I L- 4 , I L- 9 , I L- 1 0 ) biom ar k er s ov er t h e expression of MMP1 was evaluat ed by ordinal least squar es ( OLS) r egr ession. A p- value < 0.05 was FRQVLGHUHG VWDWLVWLFDOO\ VLJQL¿FDQW $OO WHVWV ZHUH perform ed in St at a14 ( St at aCorp LP College St at ion, Texas, USA) or GraphPad Prism 6.05 ( GraphPad Soft ware, I nc, San Diego, California, USA) .
RESULTS
M M P1 - 1 6 0 7 SN P fr e qu e n cy a n a lysis
The subj ect sam ple included in t his st udy was sim ilarly com posed by m ale and fem ale subj ect s ( Tab l e 1 ) . Th e f r eq u en cy o f MMP1 - 1 6 0 7 SNP genot ypes and alleles in cont rol group was sim ilar t o t hat previously report ed for t he Brazilian populat ion24
an d t h e Ch i- squ ar e t est f or t h e dist r ibu t ion of gen ot y pes in pat ien t s an d con t r ols r en der ed a p-value of 0.469 and 0.243, respect ively, which was com pat ible w it h t he Hardy- Weinberg equilibrium . When t he pat ient s pr esent ing per iapical lesions w ere com pared w it h t he cont rols, it was observed t h at t h e f r eq u en cy of MMP1 - 1 6 0 7 1 G/ 2 G w as higher in t he pat ient s group ( p= 0.0110, OR= 2.143, Cl= 1.209 t o 3.799) , as w ell t he com bined m ut ant genot y pes 1G/ 2G+ 2G/ 2G was m or e fr equent in t he pat ient s ( p= 0.0326, OR= 1.764, Cl= 1.047 t o 2.971) group t han in cont rols ( Table 1) . When t he fr equency of t he alleles was com par ed bet w een t he pat ient s and cont rols groups, no differences w ere found. The frequency of t he MMP1- 1607 SNP
genot ypes and alleles was also com pared w it hin pat ient ’s present ing act ive and inact ive periapical OHVLRQV KRZHYHU QR VLJQL¿FDQW YDULDWLRQV ZHUH found in t he frequencies ( Table 2) .
Associat ion bet w een MMP1 - 1 6 0 7 w it h MMP-1 m RN A e x pr e ssion
Ou r d a t a i n i t i a l l y sh o w e d a w e a k MMP- 1 expression in healt hy periapical t issues sam pled IURPFRQWUROVXEMHFWVZKLOHDVLJQL¿FDQWO\VWURQJHU expression was evidenced in t he periapical lesions har vest ed fr om pat ient s ( Figur e 1) . I n addit ion, our dat a dem onst rat e t hat MMP- 1 m RNA levels ZHUHVLJQL¿FDQWO\KLJKHULQDFWLYHWKDQLQLQDFWLYH lesions ( Figur e 1) . When analy zing t he possible associat ion bet w een MMP- 1 m RNA levels and t he MMP1- 1607 SNP genot ypes in cont rol group, w e found t hat polym orphic allele 2G ( 1G/ 2G+ 2G/ 2G JHQRW\SHVFDUULHUVSUHVHQWHGDVLJQL¿FDQWO\KLJKHU MMP- 1 m RNA expression w hen com pared w it h t he 1G/ 1G genot ype group ( Figure 1) . Sim ilar result s w ere observed w it hin t he periapical lesions, w here t he carriers of polym orphic allele 2G ( 1G/ 2G+ 2G/ 2G JHQRW\SHVFDUULHUVSUHVHQWHGDVLJQL¿FDQWO\KLJKHU MMP- 1 m RNA ex pr ession t han 1G/ 1G genot y pe car r ier s ( Figur e 1) . When t he per iapical lesions w e r e e v a l u a t e d a f t e r st r a t i f i ca t i o n b y l e si o n act ivit y/ inact ivit y st at us, it was observed t hat t he expression of MMP- 1 was higher in 1G/ 2G+ 2G/ 2G versus 1G/ 1G genot ypes in bot h subgroups ( Figure 1) . Addit ionally, t he or der ed logist ic r egr ession SRLQWHG WR D VLJQL¿FDQW LQÀXHQFH RI WKH 003 1607 SNP over t he m RNA MMP- 1 expression levels, dem onst rat ed by a likelihood rat io ( LR) chi- squared of 6.09 ( probabilit y of > chi- squared= 0.0136) .
Table 1- Frequencies of MMP1-1607 SNP in control subjects and patients presenting periapical lesions
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Control (N=214) Lesions (N=111) p value
N and gender distribution 103 f / 111 m 87 f / 91 m 0.8398*
MMP1-1607 genotypes C (N=214) L (N=111) p value*
1G/1G 75 (35.04) 26 (23.42)
1G/2G 70 (32.71) 52 (46.84) p=0.0110 OR=2.143
Cl=1.209 to 3.799
2G/2G 69 (32.24) 33 (29.72) p= 0.3542 OR=1.380
Cl= 0.7501 to 2.537
1G/2G + 2G/2G 139 (64.95) 85 (76.57) p= 0.0326 OR=1.764
Cl=1.047 to 2.971
MMP1-1607 alleles C (n=428) CP (n=222)
1G 220 (51.40) 104 (46.84)
2G 208 (48.59) 118 (53.15) p= 0.2830 OR=1.200
Active (N=40) Inactive (N=71) p value
MMP1-1607 genotypes A (N=40) I (N=71) p value*
1G/1G 9 (22.50) 17 (23.94)
1G/2G 17 (42.50) 35 (49.29) p=1.0000 OR= 1.090
Cl= 0.4032 to 2.947
2G/2G 14 (35.00) 19 (26.76) p=0.5988 OR=0.7185
Cl=0.2482 to 2.080
1G/2G + 2G/2G 31 (77.50) 54 (76.05) p=1.0000 OR=0.9222
Cl= 0.3671 to 2.316
MMP1-1607 alleles C (n=83) CP (n=142)
1G 38 (45.78) 69 (48.59)
2G 45 (54.21) 73 (51.40 p=0.7821 OR=0.8934
Cl= 0.5190 to 1.538 Table 2- Frequencies of MMP1-1607 SNP in patients presenting active and inactive periapical lesions
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Figure 1- MMP1-1607 SNP and its association with MMP-1 mRNA levels in periapical tissues. Total RNA was extracted from healthy periodontal ligament (control, N=26) and periapical granulomas (lesions, N=111) and levels of MMP-1 mRNA were measured quantitatively by RealTimePCR using TaqMan chemistry; the results are presented as expression of the individual mRNAs (with normalization to beta-actin using the Ct method). A) MMP-1 expression in healthy periapical tissues and periapical lesions; B) MMP-1 expression in healthy periapical tissues and periapical lesions categorized into
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and periapical lesions patients according to their genotype for MMP1-1607, determined by RealTimePCR using Taqman
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OPG expression according to patients genotype for MMP1-1607, determined by RealTimePCR using Taqman chemistry.
Asso ci a t i o n b e t w e e n M M P - 1 a n d m RN A e x pr e ssion of cyt ok in e s
Cy t ok in es an d T h elp er m ar k er s p r ev iou sly associat ed w it h p er iap ical lesion s act iv it y an d inact ivit y clust ers were correlat ed wit h MMP- 1 m RNA t ranscr ipt s t o ident ify possible associat ions. The FRUUHODWLRQFRHI¿FLHQWVEHWZHHQ003H[SUHVVLRQ
and ex pr ession levels of cy t ok ines w er e: TNF-D 0.3812, p< 0.0001; I L- 21 0.3141, p= 0.0008; I L- 17A S ,)1DŽS)2;S - 0.1581, p= 0.09; I L- 10 - 0.3166, p- = 0.0008; I L- 9 - 0.4376, p< 0.0001; I L- 4 - 3041, p= 0.0012. Our result s dem onst rat ed t hat t he cyt okines associat ed wit h lesion act ivit y clust ers, nam ely TNF-D, I L- 21, I L- 17A, and I FN- g w ere posit ively correlat ed w it h MMP- 1 levels ( Figur e 2) , w hile I L- 10, I L- 9, and I L- 22, cyt okines associat ed wit h lesions inact ivit y, p r e se n t e d n e g a t i v e co r r e l a t i o n s w i t h MMP- 1 WUDQVFULSWV )LJXUH 1R VLJQL¿FDQW FRUUHODWLRQV w ere observed bet w een t he levels of MMP- 1 and regulat ory T cell m arker FOXp3 ( Figure 2) .
Re gr e ssion a n a lysis
Si n ce MMP1 - 1 6 0 7 a n d m u l t i p l e cy t o k i n e s w er e found t o be associat ed w it h MMP- 1 m RNA l ev el s i n p er i ap i cal l esi o n s, w e su b seq u en t l y perform ed a OLS regression analysis t o m odel t he relat ive cont ribut ion of t he MMP1- 1607 SNP and each biom ar ker t o t he overall MMP- 1 r egulat ion 7DEOH 7KH ¿UVW PRGHOLQWHJUDWHGWKHJHQHWLF poly m or phism and t he ex pr ession lev els of t he SURLQÀDPPDWRU\F\WRNLQHV71)D, I L- 21, I L- 17A, DQG,)1DŽDVH[SODQDWRU\YDULDEOHV7KH5VTXDUH was 0.4044, p< 0.0001 ( adj ust ed R- square 0.3757) , ZLWKWKHSDUDPHWHUHVWLPDWHFRHI¿FLHQWVIRU MMP1- 1607 polym orphism ( p= 0.025) ; 0.3897 for ,/$S IRU,)1DŽS 0.2629 for TNF-D ( p= 0.031) , and 0.1889 for I
L-&RHI¿FLHQW Std. error P>/t/
FoxP3 -0.0377 0.1424 0.791
IL-10 -0.2552 0.1184 0.033
IL-9 -0.6067 0.1501
IL-4 -0.4059 0.1564 0.011
TNF-D 0.2629 0.1201 0.031
IL-21 0.1889 0.085 0.028
IL-17A 0.3897 0.1088 0.001
,)1Ȗ 0.3261 0.0808
Table 3- 2/6 UHJUHVVLRQ FRHI¿FLHQWV VWDQGDUG HUURU and p-value of 2-tailed t-static (null hypothesis that the
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amount of change in the dependent variable that a unit change in each of the explanatory variables produces, holding all other variables constant. p>/t/ represents the two-tailed p-values used in testing the null hypothesis that
WKHUHVSHFWLYHFRHI¿FLHQWLVHTXDOWR]HURKDVQRHIIHFWLQ
the dependent variable)
Figure 2- Cytokines, T helper markers and their association with MMP-1 mRNA levels in periapical lesions. Total RNA was extracted from periapical granulomas (N=111) and levels of MMP-1, TNF-D, IL-21, IL17A, IFNg, FOXp3, IL-10, IL-9, and IL-4 mRNA were measured quantitatively by RealTimePCR using TaqMan chemistry (with normalization to beta-actin using the Ct method). The graphs depict the linear correlation of MMP-1 and the other markers expression levels periapical
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21 ( p= 0.028) . The second m odel int egrat ed t he genet ic poly m or phism and t he ex pr ession lev el RI WKH DQWLLQÀDPPDWRU\ ELRPDUNHUV )R[3 ,/ 4, I L- 9, and I L- 10 as explanat ory variables. The R- square was 0.3253, p< 0.0001 ( adj ust ed R- square ZLWKWKHSDUDPHWHUHVWLPDWHFRHI¿FLHQWV - 0.6067 for I L- 9 ( p< 0.0001) ; 0.5181 for MMP1-1607 poly m or phism ( p> 0. 05) ; - 0. 4059 for I L- 4 ( p = 0 . 0 1 1 ) ; - 0 . 2 5 5 2 f or I L- 1 0 ( p = 0 . 0 3 3 ) ; an d - 0.0377 for FoxP3 ( p> 0.05) . For each m odel, t he R- square represent ed t he proport ion of t he variance of MMP- 1 expression at t ribut ed t o t he variat ion of t he explanat ory variables ( adj ust ed by t he num ber of var iables in t he case of adj ust ed R- squar e) . 7KHVLJQL¿FDQWFRHI¿FLHQWVUHSUHVHQWWKHYDOXHRI t he regression equat ion for predict ing t he MMP- 1 expression from t he explanat ory variable, holding all ot her variables const ant . For exam ple, t he I L- 9 FRHI¿FLHQWRISSUHGLFWVWKDWIRU every unit increase in I L- 9 w e expect a - 0.6067 decrease in MMP- 1 expression, holding all ot her variables const ant ( Table 3 and Figure 3) .
D I SCUSSI ON
The increased expression of prot eolyt ic enzym es, such as MMP- 1, is supposed t o cont ribut e t o t he on set an d pr ogr ession of per iapical lesion s by prom ot ing t he degradat ion of soft and m ineralized t issues surrounding t he t oot h apex. I n t his cont ext , WKH LGHQWL¿FDWLRQ RI WKH IDFWRUV WKDW GHWHUPLQH
t h e con t r ol of MMP- 1 ex p r ession in p er iap ical environm ent m ay com prise im port ant inform at ion t o p r ed ict lesion s d ev elop m en t or t o d ev elop t herapeut ic st rat egies t o lim it lesion’s progression.
Our r esult s init ially dem onst rat e t hat MMP- 1 m RNA levels w ere increased in periapical lesions w hen com pared w it h healt hy periapical t issues, in accordance w it h previous report s1,31. I n addit ion,
DFWLYH SHULDSLFDO OHVLRQV SUHVHQW D VLJQL¿FDQWO\ higher MMP- 1 ex pr ession t han inact iv e lesions, r ein f or cin g t h e h y p ot h esis t h at MMP- 1 in f act cont ribut e t o lesions m aint enance or progression. Accor dingly, pr ev ious st udies dem onst rat e t hat act iv e lesion s pr esen t a r edu ced ex pr ession of h ealin g m ar k er s w h en com par ed w it h in act iv e RQHV DV ZHOO DV GLVWLQFW F\WRNLQH SUR¿OHV15, 23. I t
is im port ant t o highlight t hat t he overall MMP- 1 ex pr ession in t he per iapical lesions, despit e it s act ivit y st at us, was evident ly variable. Therefore, our next st ep was t o invest igat e if a genet ic variat ion in t he MMP- 1 gene prom ot er, nam ely MMP1- 1607 SNP, previously described as a regulat or of MMP- 1 expression levels29, could account for t he MMP- 1
m RNA m odulat ion in periapical lesions as w ell as for an increased risk for lesions developm ent .
Regarding t he genet ic associat ion point of view, ou r r esu lt s dem on st r at e a h igh er fr equ en cy of
t he 1G/ 2G genot ype, as w ell as of t he com bined 1 G/ 2 G+ 2 G/ 2 G p oly m or p h ic g en ot y p es, in t h e subj ect s present ing periapical lesions t han in t he cont r ol gr oup. Ther efor e, such genot ypes add a VLJQL¿FDQW ULVN IRU WKH LQGLYLGXDO VXVFHSWLELOLW\ WR develop periapical lesions. I nt erest ingly, a previous st udy dem onst rat e t he associat ion of MMP- 2 and MMP- 3 variant s w it h periapical lesion form at ion in individuals w it h unt reat ed deep carious lesions26,
suggest ing t hat indiv iduals genet ically pr one t o increased product ion of prot eolyt ic enzym es m ay experience an increased risk for periapical lesions. I t is im port ant t o consider t hat t he increased risk obser ved in t his st udy m ay be under est im at ed, since t he cont r ol populat ion does not com pr ise individuals st rict ly exposed t o t he m aj or risk fact ors f or p er iap ical lesion s su ch as u n t r eat ed d eep carious lesions9,26. While t he select ion of different
cont rol populat ions m ay in fact im pact t he pow er and odds of genet ic st udies16WKHLGHQWL¿FDWLRQRI
a given risk fact or, such as MMP1- 1607 SNP, in a case- cont rol set t ing t hat t heoret ically m inim izes t he changes of associat ion, reinforces t hat t his fact or in fact com prises a risk elem ent9,15,26. How ever, no
differences w ere observed in t he frequency of t he MMP1- 1607 genot ypes and alleles in t he pat ient s present ing act ive or inact ive lesions; how ever, t he lim it ed pow er of such fragm ent ed analysis due t o t he dilut ion of t he experim ent al sam ple w it hin t w o VXEJURXSVGRQRWDOORZGH¿QLWLYHFRQFOXVLRQVLQWKLV VSHFL¿FDQDO\VLV
Af t er t h e i d en t i f i cat i o n o f p o si t i v e g en et i c associat ion of MMP1- 1607 w it h periapical lesion’s r i sk , w e n e x t e v a l u a t e d f r o m t h e f u n ct i o n a l view point if t he MMP- 1 genet ic variant s w ere in fact associat ed w it h MMP- 1 t ranscript s levels in t he lesions. I n fact , our result s dem onst rat e t hat t he MMP1- 1607 2G allele was associat ed wit h increased MMP- 1 m RNA expression in bot h healt hy periapical t issues and in t he periapical lesions. Accordingly, such poly m or phism was pr ev iously descr ibed t o affect gene t ranscript ion in vit ro, since t he 2G allele, t oget her wit h an adj acent adenosine, creat es a core binding sit e ( 5´ - GGA- 3´ ) for t ranscript ion fact ors im m ediat ely adj acent t o an AP- 1 sit e, causing a VLJQL¿FDQWLQFUHDVHLQWKHWUDQVFULSWLRQDFWLYLW\22.
in cr em en t ally all ot h er ex p lan at or y v ar iab les) point s t o a cont ribut ion ranging bet w een 2 and 5% ( dat a not shown) . This seem ingly weak effect m ust be int erpret ed carefully, since OLS m odels are a VLPSOL¿FDWLRQRIFRPSOH[SKHQRPHQDSDUWLFXODUO\LQ t he cont ext of biological processes. More im port ant t han t he com put ed pr edict iv e value is t he fact t hat t he genet ic poly m or phism dem onst rat ed a VLJQL¿FDQW SUHGLFWLYH HIIHFW RQ WKH RYHUZKHOPLQJ m aj orit y of t he regressions com put ed.
I t is com pu lsor y t o con sider t h at n u m er ou s SNPs are in fact supposed t o cont ribut e t o a given RXWFRPH ZLWK D VLJQL¿FDQW EXW UHODWLYHO\ VPDOO biological effect . I nt erest ingly, in a previous st udy ou r g r ou p d escr ib ed t h e associat ion b et w een MMP1- 1607 and t he MMP- 1 m RNA levels in healt hy periodont al t issues, while in periodont al lesions t he SUHVHQFH RI PLFURELDO DQG LQÀDPPDWRU\ VWLPXOL seem s t o overcom e t he genet ic predisposit ion t o higher MMP- 129. I n t his cont ext , since periapical
OHVLRQV DUH SOHQW\ RI LQÀDPPDWRU\ VLJQDOV ZH QH[W LQYHVWLJDWHG LI SURLQÀDPPDWRU\ F\WRNLQHV p r ev i o u sl y a sso ci a t ed w i t h p er i a p i ca l l esi o n s act iv it y3 cou ld con t r ibu t e t o t h e m odu lat ion of
MMP- 1 expression levels. Our result s dem onst rat e t hat t he cyt okines associat ed w it h lesion act ivit y clust ers, nam ely TNF-D, I L- 21, I L- 17A, and I FN- g w ere posit ively correlat ed w it h MMP- 1 levels upon individual linear regression analyzes. Accordingly, all t his cyt okines were previously described as posit ive regulat ors of MMP- 1 expression in different m odels8.
When t he OLS regression analysis was applied t o consider t he occurrence of t he SNP and t he level of expression of cyt okines sim ult aneously, I L- 17A and ,)1DŽZHUHUDQNHGDVWKHPDMRUGHWHUPLQDQWVRI 003OHYHOVLQSHULDSLFDOOHVLRQVZLWKFRHI¿FLHQWV of 0.3897 and 0.3261, respect ively. I nt erest ingly, KLJKOHYHOVRI,/$DQG,)1DŽZHUHREVHUYHGLQ t he cyt okine clust er associat ed w it h t he highest degree of periapical lesions act ivit y3, but w ere also
individually im plicat ed w it h lesions’ act ivit y in t he clust ers ranked in 2 and 3 posit ions3, reinforcing
t hat t heir involvem ent in lesions’ progression m ay in fact inv olv e t he upr egulat ion of MMP- 1. The OLS r egr ession analy sis also dem onst rat ed t hat 71)Į DQG ,/ VLJQL¿FDQWO\ DFFRXQW IRU 003 UHJXODWLRQZLWKFRHI¿FLHQWVRIDQG UHVSHFWLYHO\ ,Q DFFRUGDQFH ERWK 71)Į DQG ,/ 21 w er e descr ibed as com ponent s of t he t op 3 ranked cyt okine clust ers associat ed w it h periapical lesions act ivit y3. I t is m andat ory t o m ent ion t hat
t he OLS regression com put ed sim ult aneously t he explanat ory effect of t he cyt okines and t he MMP1-1607 SNP over t he overall r egulat ion of MMP- 1 levels in periapical lesions.
On t he ot her hand, t he cyt okines prevalent in clust ers associat ed w it h lesions inact ivit y, nam ely I L- 10, I L- 9, and I L- 43, w ere found t o be inversely
correlat ed w it h MMP- 1 levels in periapical lesions u p o n i n d i v i d u a l l i n e a r r e g r e ssi o n a n a l y ze s. Sim ilarly, t he OLS regression analysis dem onst rat ed WKDW,/,/DQG,/VLJQL¿FDQWO\DFFRXQWIRU GRZQUHJXODWLQJ 003 P51$ OHYHOV FRHI¿FLHQWV - 0 . 2 5 5 2 , - 0 . 6 0 6 7 , an d - 0 . 4 0 5 9 , r esp ect iv ely ) . While som e st udies dem onst rat e t hat I L- 10 can block MMPs product ion ( especially MT1- MMP, MMP-1, and MMP- 9) by t um oral cells33, t her e ar e no
direct evidences of t he direct inhibit ion of MMP- 1 by I L- 10, I L- 9, or I L- 4. How ever, it is possible t o hypot hesize t hat t he negat ive correlat ion is derived IURPDQLQGLUHFWHIIHFWVLQFHWKHDQWLLQÀDPPDWRU\ propert ies of such cyt okines, especially t he m ost st udied I L- 10, result in t he dow nregulat ion of t he cyt okines responsible for MMP- 1 upregulat ion, such as TNF-D,/,/$DQG,)1DŽ27.
I t is also in t er est in g t o ex plor e t h e lack of correlat ion bet w een FOXp3 and MMP- 1 expression i n b o t h l i n e a r a n d OLS r e g r e ssi o n a n a l y si s. FOXp3 is t h e pr ot ot y pic t r an scr ipt ion f act or of Tr egs ( r egulat or y T cells) , a T cell subset w it h pot ent im m unoregulat ory propert ies, described t o at t enuat e experim ent al periodont al and periapical lesions progression in vivo11,17. I n hum an periapical
lesions, t he ex pr ession of FOXp3 as w ell as it s m et hylat ion levels are indicat ors of lesions inact ivit y st at u s2. I n act iv e p er iap ical lesion s, in cr eased
FOXp3 m et hylat ion was associat ed w it h reduced FOXp3 m RNA expression, as w ell as reduced levels of I L- 10, a charact erist ic im m unoregulat ory product of Tr egs5. How ev er, since FOXp3 is a r egulat or
of Tr egs biology, w hich in t ur n ar e supposed t o PRGXODWH OHVLRQV¶ LQÀDPPDWRU\ PLOLHX YLD ,/ ( as w ell as via ot her im m unoregulat ory m olecules su ch as TGF- b an d CTLA- 4 ) , t h e ex ist en ce of m ult iple elem ent s in t he im m unoregulat ory cascade bet w een FOXp3 and MMP- 1 ( i.e., in a hypot het ical ov er si m p l i f i ed p at h w ay, FOXp 3 r eg u l at es t h e d ev el o p m en t o f Tr eg s, w h i ch p r o d u ce I L- 1 0 , w h ich in t u r n can d ow n r eg u lat e t h e lev els of WKH LQÀDPPDWRU\ F\WRNLQHV WKDW GLUHFWO\ FRQWURO MMP- 1 expr ession)5 m ay account for t he lack of
correlat ion bet w een such fact ors. I ndeed, w hile in vivo evidences link Tregs w it h t he m odulat ion of event s coordinat ed by MMPs and TI MPs, such as WLVVXH UHPRGHOLQJ GHJUDGDWLRQ DQG ¿EURVLV WKH current evidences point s t o an indirect regulat ion of MMPs/ TI MPs balance der iv ed fr om cy t ok ines produced and regulat ed by Tregs inst ead of a direct r egu lat ion1 7. Addit ion ally, it is n ot ew or t hy t h at
com put ed r egr ession m odels fail t o capt ur e t he subt le and int erm ingled regulat ory int erdependence bet ween m ult iple explanat ory variables and t hat t he sim ult aneous inclusion of ordinal and cont inuous dat a in t he OLS t ends t o underest im at e t he relat ive cont ribut ion of t he cont inuous variables.
m ult iple host fact ors seem t o cont ribut e t o t he local m odulat ion of MMP- 1 levels in periapical lesions. Th e MMP1 - 1 6 0 7 SNP w as dem on st r at ed t o be funct ional; being t he 2G genot ypes associat ed wit h LQFUHDVHG003P51$OHYHOVDVZHOODVLGHQWL¿HG as a risk fact or for periapical lesions developm ent in t he genot ypic case- cont rol associat ion analysis. I n addit ion, t he cyt okines TNF-D, I L- 21, I L- 17A, and ,)1DŽZHUHIRXQGWREHDVVRFLDWHGZLWKLQFUHDVHG MMP- 1 lev els in per iapical lesions, w hile I L- 10, I L- 9, or I L- 22 pr esent ed an inver se associat ion. St ill, furt her cause- and- effect st udies are required t o dissect t he int racellular net w or k s involved in t he regulat ion of MMP- 1 levels upon m ult iple and GLYHUJHQW SUR DQG DQWLLQÀDPPDWRU\ VWLPXOL LQ d if f er en t g en et ic b ack g r ou n d s scen ar ios. Su ch st udies m ay support ( or discard) t he applicat ion of MMP1- 1607 SNP as biom arker for periapical lesions r isk , as w ell t o dev elop st rat egies t o m odulat e MMP- 1 levels in t he clinical pract ice, and t herefore cont r ibut e t o im pr ove t he diagnosis and clinical m anagem ent of t hese pat hologies.
CON CLUSI ON
7KH 003 613 ZDV LGHQWL¿HG DV D ULVN fact or for periapical lesions developm ent as w ell as t o be associat ed w it h increased MMP- 1 m RNA expression in periapical t issues and lesions. The expression of MMP- 1 is also under t he cont rol of WKH SUR DQG DQWLLQÀDPPDWRU\ PLOLHX HOHPHQWV being t he cyt okines TNF-D, I L- 21, I L- 17A, and I FN- g associat ed wit h increased MMP- 1 levels in periapical lesion s, w h ile I L- 1 0 , I L- 9 , or I L- 4 pr esen t ed a negat ive correlat ion w it h MMP- 1 expression.
ACKN OW LED GM EN TS
The aut hors w ould like t o t hank Daniele Ceolin, Pat ricia Germ ino, Tania Cest ari, and Tiago Dionísio for t heir excellent t echnical assist ance. This st udy was suppor t ed by scholar ships and grant s fr om FAPESP - São Paulo Research Foundat ion and CNPq
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Developm ent 7KH DXWKRUV GHQ\ DQ\ FRQÀLFWV RI
int erest relat ed t o t his st udy.
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