Decrease in matrix metalloproteinase-3 activity in systemic sclerosis fibroblasts causes α2-antiplasmin and extracellular matrix deposition, and contributes to fibrosis development( 本文 (Fulltext) )

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Title

Decrease in matrix metalloproteinase-3 activity in systemic sclerosis fibroblasts causes α2-antiplasmin and extracellular matrix deposition, and contributes to fibrosis development( 本文 (Fulltext) )

Author(s) 丹羽, 宏文

Report No.(Doctoral

Degree) 博士(医学) 甲第1158号

Issue Date 2021-03-25

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/81567

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

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Abstract. Systemic sclerosis (SSc) is a connective tissue disease RIDXWRLPPXQHRULJLQFKDUDFWHUL]HGE\ÀEURVLVRIWKHVNLQ and visceral organs, and peripheral circulatory disturbance.

Ơ2-antiplasmin (Ơ2AP) is the major circulating inhibitor RISODVPLQDQGLVDNH\UHJXODWRURIÀEULQRO\VLV,WKDVEHHQ demonstrated that the expression of Ơ2AP is elevated in dermal ÀEUREODVWVREWDLQHGIURPSDWLHQWVZLWK66FSDWLHQWV,WKDVDOVR been determined that Ơ$3LVDVVRFLDWHGZLWKWKHGHYHORSPHQW DQGSURJUHVVLRQRIÀEURVLVLQ66F7KHSUHVHQWVWXG\DVVHVVHG WKHUHODWLRQVKLSEHWZHHQƠ2AP and matrix metalloproteinase-3 (MMP-3), an extracellular matrix (ECM)-degrading enzyme.

Serum levels of Ơ$3DQG003ZHUHPHDVXUHGLQKHDOWK\

FRQWUROVDQGSDWLHQWVZLWK66FXVLQJ(/,6$1RVLJQLÀFDQW GLIIHUHQFHV ZHUH GHWHUPLQHG EHWZHHQ WKHVH WZR JURXSV Ơ2AP, MMP-3 and tissue inhibitor of metalloproteinase-1 7,03H[SUHVVLRQZDVVXEVHTXHQWO\HYDOXDWHGLQQRUPDO DQG66FÀEUREODVWVYLDZHVWHUQEORWWLQJ7KHUHVXOWVUHYHDOHG that Ơ$3H[SUHVVLRQLQFUHDVHGLQ66FGHUPDOÀEUREODVWV ZKLOHWKHUDWLRRI0037,03GHFUHDVHG$GGLWLRQDOO\

incubation of recombinant Ơ$3ZLWK003FDXVHGƠ2AP degradation. The mixture of recombinant Ơ$3ZLWK003 ZDVVXEVHTXHQWO\DGGHGWRQRUPDOÀEUREODVWVSULRUWRZHVWHUQ blotting. The results revealed decreased Ơ-smooth muscle actin (Ơ60$DPDUNHURIWKHP\RÀEUREODVWSKHQRW\SHDQGW\SH, FROODJHQH[SUHVVLRQ7KHVWLPXODWLRQRI66FÀEUREODVWVZLWK

MMP-3 decreased protein levels of Ơ2AP, Ơ-SMA and type I collagen, thus reversing the pro-fibrotic phenotype of SSc ÀEUREODVWV66FÀEUREODVWWUDQVIHFWLRQZLWKPLFUR51$D resulted in a decreased TIMP-1 expression, but also decreased the protein expression of Ơ2AP. The results indicated that 003DWWHQXDWHGÀEURVLVSURJUHVVLRQE\GHJUDGLQJƠ2AP and ECM, and might therefore contribute to a novel therapeutic approach for SSc treatment.

Introduction

Systemic sclerosis (SSc) is a chronic connective tissue disease WKDW FDXVHV ZLGHVSUHDG PLFURYDVFXODU GDPDJH DQG H[FHV- VLYHFROODJHQGHSRVLWLRQLQWKHVNLQDQGLQWHUQDORUJDQV +RZHYHUWKHHWLRORJ\SDWKRJHQHVLVDQGSURJUHVVLRQRIWKLV disease are not fully understood.

Alpha2-antiplasmin (Ơ$3LVDN'DSURWHLQWKDW inactivates plasmin and thereby inhibits fibrinolysis (2,3).

Ơ$3 H[LVWV LQ YDULRXV WLVVXHV VXFK DV WKH OLYHU NLGQH\

intestine, spleen, lung, muscle, ovary, testis, cerebral cortex, KLSSRFDPSXVFHUHEHOOXPERQHVNLQDQGSODFHQWDRIPXULQH tissue (4). Apart from the inhibition of plasmin, Ơ2AP regulates various cell functions, including proliferation, GLIIHUHQWLDWLRQDQGF\WRNLQHSURGXFWLRQDQGDOVRDVVRFLDWHV ZLWK DQJLRJHQHVLV WLVVXH UHSDLU YDVFXODU UHPRGHOLQJ DQG ÀEURVLVSURJUHVVLRQ,QSDWLHQWVZLWKUKHXPDWLFGLVHDVHV including SSc, plasma levels of the plasmin-Ơ2AP complex are increased (14,15). Ơ$3DIIHFWVP\RÀEUREODVWGLIIHUHQWLDWLRQ extracellular matrix (ECM) production, vascular dysfunc- WLRQDQGSURJUHVVLRQRI66F,QDGGLWLRQZHKDYHVKRZQWKDW Ơ2AP levels are elevated in an SSc mouse model and dermal ÀEUREODVWV

Matrix metalloproteinase-3 (MMP-3) plays a pivotal role in ECM turnover as it can degrade ECM components, including SURWHRJO\FDQVFROODJHQ,,,,99DQG,;ODPLQLQÀEURQHFWLQ JHODWLQDQGHODVWLQ003LVH[SUHVVHGE\ÀEUREODVWV chondrocytes, osteoblasts, endothelial cells, smooth muscle cells and macrophages (20). Jinnin et al (21) observed similar MMP-3 serum levels in SSc patients and healthy controls;

KRZHYHUVHUXPOHYHOVRIDQWL003DXWRDQWLERG\DQGWLVVXH LQKLELWRUVRIPHWDOORSURWHLQDVH7,03ZHUHKLJKHULQ66F

Decrease in matrix metalloproteinase-3 activity in systemic VFOHURVLVÀEUREODVWVFDXVHV Ơ 2-antiplasmin and extracellular

PDWUL[GHSRVLWLRQDQGFRQWULEXWHVWRÀEURVLVGHYHORSPHQW

HIROFUMI NIWA¹, YOSUKE KANNO^1,2, EN SHU¹ and MARIKO SEISHIMA¹

1'HSDUWPHQWRI'HUPDWRORJ\*LIX8QLYHUVLW\*UDGXDWH6FKRRORI0HGLFLQH*LIX

2Department of Clinical Pathological Biochemistry, Faculty of Pharmaceutical Science, 'RVKLVKD:RPHQV&ROOHJHRI/LEHUDO$UWV.\RWR-DSDQ

Received February 7, 2020; Accepted July 1, 2020 '2,PPU

Correspondence to: 'U +LURIXPL 1LZD 'HSDUWPHQW RI Dermatology, Gifu University Graduate School of Medicine,

<DQDJLGR*LIX-DSDQ (PDLOKBQLZD#JLIXXDFMS

Abbreviations: SSc, systemic sclerosis; Ơ2AP, Ơ2-antiplasmin;

ECM, extracellular matrix; TIMP-1, tissue inhibitor of metalloproteinase-1

Key words: alpha2-antiplasmin, Systemic sclerosis, Fibrosis, matrix metallopeptidase-3, tissue inhibitor of metalloproteinase-1

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NIWA et al: THE ROLE OF MMP-3 AND Ơ2AP IN THE PATHOGENESIS OF SSC

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patients, suggesting that MMP-3 activity may be decreased in SSc (20,22). Since it has been reported that cleavage by MMP-3 inactivates Ơ2AP (23), the suppression of MMP-3 activity in SSc may promote the activation of Ơ2AP.

,Q WKH SUHVHQW VWXG\ ZH LQYHVWLJDWHG WKH UHODWLRQVKLS EHWZHHQƠ2AP and MMP-3 to gain insights into the pathogenesis of SSc.

Materials and methods

7KH H[SHULPHQWV ZLWK KXPDQ VDPSOHV LQ WKLV VWXG\ ZHUH approved by the Gifu University Graduate School of Medicine (WKLFV&RPPLWWHH$SSURYHG,':HUHFHLYHGZULWWHQ informed consent from the patients and volunteers involved.

Cell culture. Human normal and SSc dermal fibroblasts ZHUHREWDLQHGIURPVHYHQSDWLHQWVZLWK66FDQGIRXUKHDOWK\

FRQWUROVDVSUHYLRXVO\GHVFULEHG)LEUREODVWVZHUHVHHGHG onto 60-mm diameter dishes and cultured in 2 ml Dulbecco's PRGLÀHG(DJOHVPHGLXP'0(0FRQWDLQLQJIHWDOFDOI VHUXP)&6DWÝ&LQDKXPLGLÀHGDWPRVSKHUHZLWK CO2DLU$IWHUGD\VWKHPHGLXPZDVUHSODFHGZLWK VHUXPIUHH'0(0+XPDQQRUPDOGHUPDOÀEUREODVWVZHUH stimulated by Ơ2AP, MMP-3 or mixture of Ơ2AP and MMP-3 IRUK,QRWKHUVWXGLHVKXPDQ66FGHUPDOÀEUREODVWVZHUH stimulated by MMP-3 for 24 h.

Western blot analysis. &HOOV ZHUH ZDVKHG WZLFH ZLWK FROG PBS, harvested, and then sonicated in lysis buffer containing P07ULV+&OEXIIHUS+6'67ULWRQ;

DQGDSURWHDVHLQKLELWRUFRFNWDLO5RFKH'LDJQRVWLFV*PE+

7KHVNLQVDPSOHVIURPWKHVXEMHFWVZHUHKRPRJHQL]HGDQG sonicated in the lysis buffer. The protein concentration in each O\VDWHZDVPHDVXUHGXVLQJD%&$SURWHLQDVVD\NLW3LHUFH 7KHUPR)LVKHU6FLHQWLÀF3URWHLQVLQWKHVXSHUQDWDQWZHUH VHSDUDWHGE\HOHFWURSKRUHVLVRQ6'6SRO\DFU\ODPLGHJHOV and transferred to a PVDF membrane. We detected each protein E\LQFXEDWLRQZLWKWKHUHOHYDQWSULPDU\DQWLERGLHVIROORZHGE\

horseradish peroxidase-conjugated antibodies to IgG.

Enzyme-linked immunosorbent assay (ELISA). Blood samples ZHUHREWDLQHGIURP66FSDWLHQWVDQGKHDOWK\YROXQWHHUV DQGZHUHVXEVHTXHQWO\FHQWULIXJHGIRUPLQDW[J 7KHVXSHUQDWDQWZDVWKHQFROOHFWHGDQGXVHGIRUWKHDVVD\

The serum levels of Ơ$3 DQG 003 ZHUH GHWHUPLQHG XVLQJ(/,6$NLWV+XPDQ6HUSLQ)Ơ 2-Antiplasmin (R&D Systems, MN, USA) and Human Total MMP-3 Immunoassay 5 '6\VWHPVUHVSHFWLYHO\7KHDEVRUEDQFHZDVPHDVXUHG DW QP XVLQJ DQ L0DUN 0LFURSODWH 5HDGHU %LR5DG Laboratories, Inc.).

miRNA study. 66FGHUPDOÀEUREODVWVZHUHWUDQVIHFWHGZLWK PL5DVHTXHQFH$&8*$888&8888**8*88&$*

Bioneer) or negative control miRNA using Lipofectamine ,QYLWURJHQ7KHUPR)LVKHU6FLHQWLÀFDFFRUGLQJWRWKH PDQXIDFWXUHUVLQVWUXFWLRQV&HOOVZHUHKDUYHVWHGKDIWHU transfection for further analysis.

Statistical analysis. $OOGDWDZHUHH[SUHVVHGDVPHDQ6(0 and analyzed using Statmate III version 3.06 (ATMS Co., Ltd.).

7KHVWDWLVWLFDODQDO\VLVZDVFRQGXFWHGZLWKXQSDLUHGWWHVWIRU WZRJURXSFRPSDULVRQVZLWKRQHZD\$129$7XNH\VIRU PXOWLSOH FRPSDULVRQ 3 ZDV FRQVLGHUHG WR LQGLFDWH D VWDWLVWLFDOO\VLJQLÀFDQWGLIIHUHQFH

Results

Serum levels of a2AP and MMP-3 are similar in healthy controls and SSc patients. We examined the levels of Ơ2AP

Figure 1. Serum levels of Ơ$3DQG003LQSDWLHQWVZLWK66FDQGKHDOWK\

FRQWUROV %ORRG VDPSOHV ZHUH REWDLQHG IURP SDWLHQWV ZLWK 66F DQG KHDOWK\YROXQWHHUVDQGVXEVHTXHQWO\FHQWULIXJHGIRUPLQDW[J DQGÝ&6XSHUQDWDQWVZHUHWKHQFROOHFWHGDQGXVHGIRUDVVHVVPHQW7KH serum levels of (A) Ơ$3DQG%003ZHUHPHDVXUHGXVLQJ(/,6$1R VLJQLÀFDQWGLIIHUHQFHVZHUHLGHQWLÀHGEHWZHHQSDWLHQWVDQGKHDOWK\FRQWUROV 'DWDDUHSUHVHQWHGDVWKHPHDQ6(0Ơ2AP, Ơ2-antiplasmin; MMP-3, matrix metallopeptidase-3; SSc, systemic sclerosis.

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Figure 2. Ơ$3H[SUHVVLRQDQG0037,03UDWLRLQKXPDQQRUPDODQG66FGHUPDOÀEUREODVWV3URWHLQOHYHOVRIƠ$3003DQG7,03ZHUHPHDVXUHG YLDZHVWHUQEORWDQDO\VLV7KHKLVWRJUDPSURYLGHVTXDQWLWDWLYHUHSUHVHQWDWLRQVRIƠ$3DQG0037,03UDWLRQRUPDOÀEUREODVWVQ 66FÀEUREODVWV Q 'DWDDUHSUHVHQWHGDVWKHPHDQ6(0^*P<0.01 and ^**P<0.05 as indicated. Ơ2AP, Ơ2-antiplasmin; MMP-3, matrix metallopeptidase-3; TIMP-1, tissue inhibitor of metalloproteinase-1; SSc, systemic sclerosis.

Figure 3. MMP-3-mediated Ơ$3GHJUDGDWLRQLQKLELWVWKHSURÀEURWLFHIIHFWVRIƠ2AP. (A) Recombinant Ơ$3ZDVLQFXEDWHGZLWKRUZLWKRXW003IRU KDWÝ&DIWHUZKLFKOHYHOVRIƠ$3ZHUHH[DPLQHGYLDZHVWHUQEORWWLQJ%5HFRPELQDQWƠ$3ZDVLQFXEDWHGZLWKRUZLWKRXW003LQDWXEHIRUK DWÝ&1RUPDOKXPDQGHUPDOÀEUREODVWVZHUHWKHQVWLPXODWHGIRUK7KHH[SUHVVLRQRIHDFKSURWHLQZDVH[DPLQHGYLDZHVWHUQEORWWLQJ7KHKLVWRJUDP SURYLGHVTXDQWLWDWLYHUHSUHVHQWDWLRQVRIHDFKSURWHLQQ 'DWDDUHSUHVHQWHGDVWKHPHDQ6(0^*P<0.01 as indicated. MMP-3, matrix metallopeptidase-3;

Ơ2AP, Ơ2-antiplasmin; ƠSMA, ƠVPRRWKPXVFOHDFWLQ)%ÀEUREODVW

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3004

)LJXUH003UHYHUVHVWKHSURÀEURWLFSKHQRW\SHRI66FGHUPDOÀEUREODVWV+XPDQ66FGHUPDOÀEUREODVWVZHUHVWLPXODWHGE\003QJPOIRUK 7KHH[SUHVVLRQRIHDFKSURWHLQZDVVXEVHTXHQWO\GHWHUPLQHGYLDZHVWHUQEORWWLQJ7KHKLVWRJUDPSURYLGHVTXDQWLWDWLYHUHSUHVHQWDWLRQVRIHDFKSURWHLQQ 'DWDDUHSUHVHQWHGDVWKHPHDQ6(0^**P<0.05 as indicated. MMP-3, matrix metallopeptidase-3; SSc, systemic sclerosis; Ơ2AP, Ơ2-antiplasmin; ƠSMA, Ơ VPRRWKPXVFOHDFWLQ)%ÀEUREODVW

)LJXUHPL51$DDWWHQXDWHVƠ$3GHSRVLWLRQLQ66FGHUPDOÀEUREODVWVE\VXSSUHVVLQJ7,03H[SUHVVLRQ+XPDQ66FGHUPDOÀEUREODVWVZHUHWUDQV- IHFWHGZLWKFRQWURORUPL51$DDQGKDUYHVWHGKDIWHUWUDQVIHFWLRQ7KHH[SUHVVLRQRIHDFKSURWHLQZDVGHWHUPLQHGYLDZHVWHUQEORWWLQJ7KHKLVWRJUDP SURYLGHVTXDQWLWDWLYHUHSUHVHQWDWLRQVRIHDFKSURWHLQQ 'DWDDUHSUHVHQWHGDVWKHPHDQ6(0^**P<0.05 as indicated. miRNA or miR, microRNA; Ơ2AP, Ơ2-antiplasmin; SSc, systemic sclerosis; TIMP-1, tissue inhibitor of metalloproteinase-1; ƠSMA, Ơ smooth muscle actin.

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and MMP-3 in the sera from healthy controls and SSc patients by ELISA and found no significant differences (healthy FRQWUROVQ 66FSDWLHQWVQ )LJ

Ơ2AP expression increased while MMP-3/TIMP-1 ratio GHFUHDVHGLQ66FGHUPDOÀEUREODVWVBonaventura et al (24) UHSRUWHG WKDW WKH UDWLR RI 0037,03 LQGLFDWHV WKH DFWLYLW\RI003+HQFHZHQH[WDVVHVVHGƠ2AP expression DQG0037,03UDWLRLQQRUPDOKXPDQDQG66FGHUPDO ÀEUREODVWVE\ZHVWHUQEORWWLQJQRUPDOÀEUREODVWVQ 66F ÀEUREODVWVQ $VVKRZQLQ)LJZHIRXQGWKDWƠ2AP H[SUHVVLRQLQGHUPDOÀEUREODVWVZDVLQFUHDVHGLQ66FZKHUHDV WKH0037,03UDWLRZDVUHGXFHG

003PHGLDWHGGHJUDGDWLRQLQKLELWVSURÀEURWLFHIIHFWVRI Ơ2AP. Cleavage by MMP-3 inactivates Ơ2AP (23). We thus incubated Ơ$3IRUKDWÝ&LQWKHSUHVHQFHRUDEVHQFH RI 003 DQG VKRZHG WKDW WKH HQ]\PH GHJUDGHGƠ2AP (Fig. 3A). To assess the effect of MMP-3-mediated degradation on Ơ$3ZHSHUIRUPHGWKHVDPHUHDFWLRQVDQGDGGHG WKHPL[WXUHVWRQRUPDOKXPDQÀEUREODVWV3UHLQFXEDWLRQRI Ơ$3ZLWK003OHGWRDWWHQXDWLRQRIWKHH[SUHVVLRQRI Ơ-smooth muscle actin (Ơ60$DPDUNHURIWKHP\RÀEUREODVW phenotype) and type I collagen (Fig. 3B).

MMP-3 reverses pro-fibrotic phenotype of SSc dermal ÀEUREODVWV1H[WZHVWLPXODWHG66FGHUPDOÀEUREODVWVZLWK 003WRLQYHVWLJDWHLWVDQWLÀEURWLFHIIHFW&RQVLVWHQWZLWK WKHSUHYLRXVUHVXOWVVWLPXODWLRQZLWK003GHFUHDVHGWKH expression of Ơ2AP, ƠSMA, and type I collagen (Fig. 4).

MicroRNA-29a attenuates Ơ2AP deposition in SSc dermal fibroblasts by inhibiting TIMP-1. &LHFKRPVNDet al (25) VKRZHG WKDW PL5D GHFUHDVHG 7,03 H[SUHVVLRQ DQG UHYHUVHGWKHSURÀEURWLFSKHQRW\SHRI66FGHUPDOÀEUREODVWV ,QWKHSUHVHQWVWXG\ZHFRQÀUPHGWKDWPL5DWUDQVIHFWLRQ LQ66FÀEUREODVWVFDXVHGDGHFUHDVHLQ7,03H[SUHVVLRQ LQ OLQH ZLWK WKH SUHYLRXV UHSRUW )LJ :H DOVR VKRZHG WKDWPL5DFDXVHGDGHFUHDVHLQƠ2AP expression in SSc ÀEUREODVWV)LJ

Discussion

66FFDXVHVÀEURVLVRIWKHVNLQDQGLQWHUQDORUJDQV3UHYLRXVO\

ZH VKRZHG WKDWƠ2AP induces TGF-ơ production through DGLSRVHWULJO\FHULGHOLSDVHDQGLVDVVRFLDWHGZLWKP\RÀEUR- EODVWGLIIHUHQWLDWLRQDQG(&0SURGXFWLRQ:HDOVRVKRZHG that the expression of Ơ2AP is elevated in SSc model mice DQG66FÀEUREODVWVDQGWKDWLWVLQDFWLYDWLRQDWWHQXDWHVGLVHDVH VHYHULW\LQ66FPRGHOPLFHDQG66FÀEUREODVWV7KHVH ÀQGLQJVVXJJHVWWKDWƠ2AP contributes to the development of ÀEURVLVLQ66F003DQ(&0GHJUDGLQJHQ]\PHLQDFWL- vates Ơ$3E\FOHDYLQJLWV3UR/HXSHSWLGHERQG +HUHZHIRFXVHGRQƠ2AP and MMP-3 to clarify their roles in the pathogenesis of SSc.

,QWKLVVWXG\ZHVKRZHGWKDWVHUXPOHYHOVRIƠ2AP and 003GLGQRWYDU\EHWZHHQKHDOWK\FRQWUROVDQG66FSDWLHQWV +RZHYHUFRQVLVWHQWZLWKRXUSUHYLRXVÀQGLQJVƠ2AP expres- VLRQLQ66FGHUPDOÀEUREODVWVZDVLQFUHDVHG7RGHWHUPLQH ZKHWKHU003FRQWULEXWHVWRWKHKLJKH[SUHVVLRQRIƠ2AP in 66FÀEUREODVWVZHPHDVXUHGWKHUDWLRRI0037,03DV

Figure 6. Role of MMP-3 and Ơ$3LQWKHGHYHORSPHQWRI66FÀEURVLV'HFUHDVHG003DFWLYLW\LQ66FGHUPDOÀEUREODVWVPD\SURPRWHWKHGHYHORSPHQWRI ÀEURVLVWKURXJKWKHVXSSUHVVLRQRI(&0DQGƠ2AP degradation. MMP-3, matrix metallopeptidase-3; Ơ2AP, Ơ2-antiplasmin; SSc, systemic sclerosis; TIMP-1, tissue inhibitor of metalloproteinase-1; ECM, extracellular matrix.

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3006

DQLQGLFDWLRQRI003DFWLYLW\2XUUHVXOWVVKRZHGWKDWWKLV UDWLRZDVORZLQ66FGHUPDOÀEUREODVWV7DNHQWRJHWKHUWKHVH data suggest that the decrease in MMP-3 activity might induce Ơ$3H[SUHVVLRQLQ66FÀEURWLFWLVVXH0RUHRYHUVNLQVSHFLÀF LQGXFWLRQDQGGHYHORSPHQWRIÀEURVLVPD\EHGXHWRLQFUHDVHG Ơ2AP expression and decreased MMP-3 activity in tissue but not in serum.

MMP-3 inactivates Ơ2AP by proteolytic cleavage (23).

+HUHZHFRQÀUPHGWKDW003FOHDYHGƠ$3LQWRWZRIUDJ- ments and attenuated the Ơ$3LQGXFHGSURÀEURWLFHIIHFWV VXFKDVP\RÀEUREODVWGLIIHUHQWLDWLRQDQGFROODJHQSURGXFWLRQ LQ QRUPDO ILEUREODVWV ,Q DGGLWLRQ WUHDWPHQW ZLWK 003 suppressed the pro-fibrotic response of SSc fibroblasts by UHGXFLQJWKHH[SUHVVLRQRIFROODJHQDQGP\RÀEUREODVWPDUNHUV 0RUHRYHU003LVNQRZQWRGHJUDGH(&0FRPSRQHQWV such as collagen. The decrease in MMP-3 activity in SSc dermal fibroblasts not only attenuates Ơ2AP inactivation but also promotes ECM deposition, and contributes to SSc progression (Fig. 6).

PL5DUHSUHVVHV7,03H[SUHVVLRQWKHUHE\UHYHUVLQJ WKHSURÀEURWLFSKHQRW\SHRI66FGHUPDOÀEUREODVWV,Q WKHSUHVHQWVWXG\ZHVKRZHGWKDWPL5DWUDQVIHFWHG66F ILEUREODVWV H[KLELWHG D ORZƠ2AP and TIMP-1 expression.

Collectively, our data suggest that TIMP-1-induced MMP-3 inhibition may lead to Ơ2AP deposition in SSc.

,QFRQFOXVLRQZHIRXQGWKDWWKHDFWLYLW\RI003ZDV decreased and that Ơ$3H[SUHVVLRQZDVLQFUHDVHGLQ66F ÀEUREODVWV0RUHRYHUWUHDWPHQWZLWK003RUVXSSUHVVLRQ ZLWKD003LQKLELWRU7,03UHYHUVHGWKHSURÀEURWLF SKHQRW\SHRI66FGHUPDOÀEUREODVWV2XUUHVXOWVVXJJHVWWKDW GHFUHDVHLQ003DFWLYLW\LQ66FÀEUREODVWVFDXVHVƠ2AP and ECM deposition, and contributes to the development of fibrosis. Thus, our findings might contribute to a novel therapeutic approach for treatment of SSc.

Acknowledgements Not applicable.

Funding

7KH FXUUHQW VWXG\ ZDV SDUWLDOO\ VXSSRUWHG E\ WKH 7DNHGD Science Foundation.

Availability of data and materials

7KHGDWDVHWVXVHGDQGRUDQDO\]HGGXULQJWKHFXUUHQWVWXG\DUH DYDLODEOHIURPWKHFRUUHVSRQGLQJDXWKRURQUHDVRQDEOHUHTXHVW Authors' contributions

HN, YK, ES and MS designed the current study. HN, YK and ES performed the experiments and analyzed data. HN, YK, (6DQG06ZURWHDQGHGLWHGWKHPDQXVFULSW$OODXWKRUVUHDG DQGDSSURYHGWKHÀQDOPDQXVFULSW

Ethics approval and consent to participate

The experiments utilizing human samples in the current VWXG\ ZHUH DSSURYHG E\ WKH *LIX 8QLYHUVLW\ *UDGXDWH

6FKRRORI0HGLFLQH(WKLFV&RPPLWWHHDSSURYDOQR :ULWWHQLQIRUPHGFRQVHQWZDVREWDLQHGIURPWKHSDWLHQWVDQG volunteers involved.

Patient consent for publication Not applicable.

Competing interests

The authors declare that there are no competing interests.

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8. .DQQR<.DZDVKLWD(0LQDPLGD0.DQHLZD$2NDGD.

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12. .DZDVKLWD ( .DQQR < $VD\DPD + 2NDGD . 8HVKLPD 6 Matsuo O and Matsuno H: Involvement of Ơ2-antiplasmin in GHQGULWLFJURZWKRIKLSSRFDPSDOQHXURQV-1HXURFKHP 13. +RX < 2NDGD . 2NDPRWR & 8HVKLPD 6 DQG 0DWVXR 2 Alpha2-antiplasmin is a critical regulator of angiotensin II-mediated vascular remodeling. Arterioscler. Thromb Vasc Biol 28: 1257-1262, 2008.

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18. Chen Q, Jin M, Yang F, Zhu J, Xiao Q and Zhang L: Matrix PHWDOORSURWHLQDVHV,QÁDPPDWRU\UHJXODWRUVRIFHOOEHKDYLRUV in vascular formation and remodeling. Mediators Inflamm:

Jun 12, 2013 (Epub ahead of print).

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