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BASIC RESEARCH Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats Tung-Yu Tsui, Chi-Keung Lau, Jian Ma, Gabriel Glockzin, Aiman Obed, Hans J Schlitt, Sheung-Tat Fan Tung-Yu Tsui, Chi-Keung Lau, Jian Ma, Sheung-Tat Fan, Centre for the Study of Liver Disease and Department of Surgery, the University of Hong Kong, Pokfulam, Hong Kong Tung-Yu Tsui, Gabriel Glockzin, Aiman Obed, Hans J Schlitt, Department of Surgery, University of Regensburg Medical Centre, Regensburg, Germany Correspondence to: Dr. Tung-Yu Tsui, Department of Surgery, University of Regensburg Medical Centre, Franz-Josef-Strauss- Allee 11, 93053 Regensburg, Germany. [email protected] Telephone: +49-941-9446852 Fax: +49-941-9446802 Received: 2005-09-12 Accepted: 2005-10-26 Abstract AIM: To test the hypothesis that enhancement of the activity of heme oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heme oxygense-1 in a CCl 4 -induced micronodular cir- rhosis model. METHODS: Recombinant adeno-associated viruses car- rying rat HO-1 or GFP gene were generated. 1×10 12 vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fi- brosis. RESULTS: Conditioning the rat liver with over-expres- sion of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The develop- ment of micronodular cirrhosis was significantly inhib- ited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to con- trols, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with im- proved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells (HSCs) in rAAV/ HO-1-transduced livers. CONCLUSIONS: Enhancement of HO activity in the liv- ers suppresses the development of cirrhosis. © 2006 The WJG Press. All rights reserved. Key Words: Cirrhosis; Portal hypertension; Heme oxy- genase; Gene therapy; Adeno-associated virus Tsui TY, Lau CK, Ma J, Glockzin G, Obed A, Schlitt HJ, Fan ST. Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats. World J Gastroenterol 2006; 12(13): 2016-2023 http://www.wjgnet.com/1007-9327/12/2016.asp INTRODUCTION Heme oxygenase �H�s a �a �e�m� �ng en�yme �a �H�s a �a �e�m� �ng en�yme �a c�eaves p�o�ox�dan�eme �n�o equ�mo�a� amoun�s of ca�bon monox�de �C�, b�ve�d�n/b� �ub�n, and f �ee , and f�ee and f�ee fe��ous �on [1,2] . Up �o da�e, ��ee �sofo�ms of H�ave been �den�f�ed. Among of �em, H�1 and H�2 a�e exp�essed �n �ve�s. H�2 exp�esses cons�u��ve�y �n �epa�ocy�e and func��ons as an �mpo�an� en�yme �n ca�a�y�ng �e endogenous and exogenous �eme. In con�as�, H�1 �s an �nduc�b�e fo�m of H�n �ve�s [3] . A��oug� exp�ess�on of H�1 �n �e �ve�s p�ma�y �es�c�ed �o a subpopu�a�on of Kupffe� ce��s [3,4] , ��e p�o�e�n �s exp�essed �n bo�� pa�enc�yma� and pa�cu�a�y non�pa�enc�yma� ��vece��s, and �e�efo�e �esu�s �n �g�e� en�yma�c ac�v��es of H� unde� s�ess cond�ons [4] . T�e cy�op�o�ec��ve effec�s of en�anced H� ac�v��es �n �ve� m�g� be ��oug�e mec�an�sms assoc�a�ed w�s ca�a�y�c p�oduc�s. A�� of �s ��ee ca�a�y�c p�oduc�s can con��bu�e ��e p�o�ec��ve effec�s of H[5�7] . H��1 was �nduced �n �e �ve�s w� c�� �os �s [8] . I� was ��oug�� �o be an adop��ve �esponse �o ox�da�ve s��esses, �nf �amma�o�y �nsu�s of pe�s�s�en� o� �ecu��en�ve�nju�y as we�� as �e �espons�veness �o ��e �nc�ease of �n�as�nuso�da�es�s�ance. H�1 was ma�n�y exp�essed �n Kupffe� ce��s, s�nuso�da� endo�e�a� ce��s, s�e��a�e ce��s and �n a pa� of �epa�ocy�e �n �ve�s w� c���os�s [8,9] . H��1 was a�so exp�essed �n vascu�a� sys�em ��a� m�g� con��bu�e �o ��e deve�opmen� of �emodynam�c c�anges �n �e �a�s w� c���os�s [9] . In add� �on, �g�e�eve�s of Cp�oduc�on m�g� be assoc�a�ed w�e deve�opmen� of �epa�opu�mona�y synd�ome �n pa�en�s w� c���os�s [10] . T�us, � becomes con��ove�sy w�e�e�e exp�ess�on of H��1 o� en�ancemen� of H� ac�v��es con�bu�e �o ��e pa��op�ys�o�og�ca� c�anges �n �e deve�opmen� of c���os�s, o� w�e�e� H�1 func�ons as �omeos�a��c mo�ecu�e �n con��o���ng of d�sease p�og�ess�on. To e�uc�da�e ��e �o�e of PO Box 2345, Beijing 100023, China World J Gastroenterol 2006 April 7; 12(13):2016-2023 www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327 [email protected] © 2006 The WJG Press. All rights reserved. www.wjgnet.com

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BASIC RESEARCH

Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats

Tung-Yu Tsui, Chi-Keung Lau, Jian Ma, Gabriel Glockzin, Aiman Obed, Hans J Schlitt, Sheung-Tat Fan

Tung-Yu Tsui, Chi-Keung Lau, Jian Ma, Sheung-Tat Fan, Centre for the Study of Liver Disease and Department of Surgery, the University of Hong Kong, Pokfulam, Hong KongTung-Yu Tsui, Gabriel Glockzin, Aiman Obed, Hans J Schlitt, Department of Surgery, University of Regensburg Medical Centre, Regensburg, GermanyCorrespondence to: Dr. Tung-Yu Tsui, Department of Surgery, University of Regensburg Medical Centre, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. [email protected]: +49-941-9446852 Fax: +49-941-9446802Received: 2005-09-12 Accepted: 2005-10-26

AbstractAIM: To test the hypothesis that enhancement of the activity of heme oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heme oxygense-1 in a CCl4-induced micronodular cir-rhosis model.

Methods: Recombinant adeno-associated viruses car-rying rat HO-1 or GFP gene were generated. 1×1012 vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fi-brosis.

Results: Conditioning the rat liver with over-expres-sion of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The develop-ment of micronodular cirrhosis was significantly inhib-ited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to con-trols, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with im-proved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells (HSCs) in rAAV/HO-1-transduced livers.

ConClusIons: Enhancement of HO activity in the liv-ers suppresses the development of cirrhosis.

© 2006 The WJG Press. All rights reserved.

Key Words: Cirrhosis; Portal hypertension; Heme oxy-genase; Gene therapy; Adeno-associated virus

Tsui TY, Lau CK, Ma J, Glockzin G, Obed A, Schlitt HJ, Fan ST. Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats. World J Gastroenterol 2006; 12(13): 2016-2023

http://www.wjgnet.com/1007-9327/12/2016.asp

IntroductIonHeme oxygenase �H��� �s a �a�e���m���ng en�yme ��a� �H��� �s a �a�e���m���ng en�yme ��a� c�eaves p�o�ox�dan� �eme �n�o equ�mo�a� amoun�s of ca�bon monox�de �C���, b���ve�d�n/b����ub�n, and f�ee, and f�ee and f�ee fe��ous ��on [1,2]. Up �o da�e, ���ee �sofo�ms of H� �ave been �den��f�ed. Among of ��em, H��1 and H��2 a�e exp�essed �n ��ve�s. H��2 exp�esses cons���u��ve�y �n �epa��ocy�e and func��ons as an �mpo��an� en�yme �n ca�a�y��ng ��e endogenous and exogenous �eme. In con��as�, H��1 �s an �nduc�b�e fo�m of H� �n ��ve�s [3]. A���oug� exp�es�s�on of H��1 �n ��e ��ve� �s p��ma���y �es���c�ed �o a sub�popu�a��on of Kupffe� ce��s [3,4], ��e p�o�e�n �s exp�essed �n bo�� pa�enc�yma� and pa���cu�a��y non�pa�enc�yma� ��ve� ce��s, and ��e�efo�e �esu��s �n ��g�e� en�yma��c ac��v���es of H� unde� s��ess cond���ons [4]. T�e cy�op�o�ec��ve effec�s of en�anced H� ac��v���es �n ��ve� m�g�� be ���oug� ��e mec�an�sms assoc�a�ed w��� ��s ca�a�y��c p�oduc�s. A�� of ��s ���ee ca�a�y��c p�oduc�s can con���bu�e ��e p�o�ec��ve ef�fec�s of H� [5�7].

H��1 was �nduced �n ��e ��ve�s w��� c����os�s [8]. I� was ��oug�� �o be an adop��ve �esponse �o ox�da��ve s��esses, �nf�amma�o�y �nsu��s of pe�s�s�en� o� �ecu��en� ��ve� �nju�y as we�� as ��e �espons�veness �o ��e �nc�ease of �n��as�nuso�da� �es�s�ance. H��1 was ma�n�y exp�essed �n Kupffe� ce��s, s�nuso�da� endo��e��a� ce��s, s�e��a�e ce��s and �n a pa�� of �epa�ocy�e �n ��ve�s w��� c����os�s [8,9]. H��1 was a�so exp�essed �n vascu�a� sys�em ��a� m�g�� con���b�u�e �o ��e deve�opmen� of �emodynam�c c�anges �n ��e �a�s w��� c����os�s [9]. In add���on, ��g�e� �eve�s of C� p�oduc��on m�g�� be assoc�a�ed w��� ��e deve�opmen� of �epa�opu�mona�y synd�ome �n pa��en�s w��� c����os�s [10]. T�us, �� becomes con��ove�sy w�e��e� ��e exp�ess�on of H��1 o� en�ancemen� of H� ac��v���es con���bu�e �o ��e pa��op�ys�o�og�ca� c�anges �n ��e deve�opmen� of c����o�s�s, o� w�e��e� H��1 func��ons as �omeos�a��c mo�ecu�e �n con��o���ng of d�sease p�og�ess�on. To e�uc�da�e ��e �o�e of

PO Box 2345, Beijing 100023, China World J Gastroenterol 2006 April 7; 12(13):2016-2023www.wjgnet.com World Journal of Gastroenterology ISSN [email protected] © 2006 The WJG Press. All rights reserved.

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Tsui TY et al . HO-1 suppresses liver fibrogenesis 2017

H��1 �n ��e con�ex� of �ecu��en� �epa�oce��u�a� �nju�y and ��e deve�opmen� of c����os�s, �e�e we �a�e ��e advan�ageof c����os�s, �e�e we �a�e ��e advan�agec����os�s, �e�e we �a�e ��e advan�age of adeno�assoc�a�ed v��a� vec�o� �o �nc�ease ��e ove�a�� H� en�yma��c ac��v��y of ��e ��ve� �n a s�ab�e manne� and �o �nves��ga�e ��e effec�s of H��1 �n ��e d�sease p�og�ess�on �n ca�bon �e��ac��o��de �CC�4����nduced c����os�s mode�.��nduced c����os�s mode�.�nduced c����os�s mode�.

MAtErIALS And MEtHodSGeneration of recombinant adeno-associated viral vectorss (r���sr���s�� rAAVs were produced and purified as previously described [11]. In b��ef, a fu����eng�� H��1 gene o��g�na��y c�oned f�oma fu����eng�� H��1 gene o��g�na��y c�oned f�omfu����eng�� H��1 gene o��g�na��y c�oned f�om��eng�� H��1 gene o��g�na��y c�oned f�om�eng�� H��1 gene o��g�na��y c�oned f�omH��1 gene o��g�na��y c�oned f�om gene o��g�na��y c�oned f�om ��e sp�een of a ���� �a� o� ��e �epo��e� gene ���� wasa ���� �a� o� ��e �epo��e� gene ���� was���� �a� o� ��e �epo��e� gene ���� was��e �epo��e� gene ���� was�epo��e� gene ���� was �nse��ed �n�o ��e vec�o� p�asm�d �o cons��uc� pSNAV1/���� o� pSNAV1/H��1. �AAV/���� o� �AAV/H��1 waswas gene�a�ed �n BHK�21 ce��s �Ame��can Type Cu��u�e Co��ec���on, Manassas, VA�� by ��ansfec��on of vec�o� p�asm�d and subsequen� �escue �AAVs �se�o�ype 2�� by co��nfec��on w��� recombinant HSV1-rc/∆UL2, which is essential for viron packaging. A large scale of rAAV was purified as described [12].

�nimal model and gene delivery protocolInb�ed ���� �a�s �230�250 g�� we�e pu�c�ased f�om ��eed f�om ��e f�om ��e An�ma� Ins���u�e of Med�ca� Sc�oo� Hannove�, �e�many and we�e ma�n�a�ned �n ��e �abo�a�o�y An�ma� Un�� of��e �abo�a�o�y An�ma� Un�� of�abo�a�o�y An�ma� Un�� of ��e Un�ve�s��y of Hong Kong. T�e s�udy was app�oved by ��e Comm���ee on ��e Use of ��ve An�ma�s fo� Teac��ng and Resea�c�, ��e Un�ve�s��y of Hong Kong. M�c�onodu�a� ��ve� c����os�s �n ���� �a�s was �nduced us�ng a p�o�oco� desc��bed p�ev�ous�y [13]. In b��ef, p�en�oba�b��a� sod�um (35 mg/L) was given one week before the first dose of CCl4 �n o�de� �o �nc�ease ��e sens���v��y of ��e �a� ��ve� �o CC�4 and was p�esen� �n d��n��ng wa�e� �n ��e w�o�e pe��od of��e w�o�e pe��od ofw�o�e pe��od of �nduc��on. A �o�a� of 9 doses of CC� �o�a� of 9 doses of CC��o�a� of 9 doses of CC�of 9 doses of CC�9 doses of CC�4 �0.2 m�/�g pe� w��� we�e g�ven �o ��e �a�s �n��agas���ca��y. �AAVs �1×1012v.g.�� was g�ven ���oug� ��e po��a� ve�n of �a�s a� ��e ��me pen�o�barbital was given in drinking water and �� d before the firstwas given in drinking water and �� d before the firstin drinking water and �� d before the firsting water and �� d before the first water and �� d before the first dose of CC�4.

Liver biochemistry, histology, immunohistochemistry and ELIS� Se�um samp�es we�e co��ec�ed a� ��e end po�n� of expe���men�s. T�e ac��v���es of a�an�ne am�no��ansfe�ase �A�T�� and �o�a� b����ub�n �n ��e p�asma we�e measu�ed �n ��e Depa��men� of C��n�ca� B�oc�em�s��y, ��e Un�ve�s��y of Hong Kong. ��ve� samp�es we�e snap f�o�en and s�o�ed a� �75 ℃ un��� fu���e� app��ca��ons. ��ve m�c�ome�e�s of f�o���ve m�c�ome�e�s of f�o� of f�o��en sec��ons we�e used fo� �ema�oxy��n and eos�n s�a�n�ng,and eos�n s�a�n�ng, eos�n s�a�n�ng, Masson’s ���c��ome s�a�n�ng, and �mmuno��s�oc�em�s��y., and �mmuno��s�oc�em�s��y. and �mmuno��s�oc�em�s��y. The area of fibrotic tissues in the cross section of liverss �n ��e c�oss sec��on of ��ve�s �n ��e c�oss sec��on of ��ve�s was measu�ed by ��e compu�e� sof�wa�e �Me�aMo�p� �m�ag�ng sys�em, Un�ve�sa� Imag�ng Co�po�a��on, ��A�� af�e� Masson’s trichrome, collagen 1α and fibronectin staining. A�� measu�emen�s we�e done �n a doub�e�b��nd manne�a doub�e�b��nd manne�doub�e�b��nd manne��b��nd manne�b��nd manne� w��� a� �eas� 20 a�eas/��ve� �f�b�o��c a�ea, o��g�na� 200×�� �n ��e 5 ��ve�s/g�oup. Mouse an����a� �D1 ��nf����a��ng mac�op�age��, an����D2 ���ssue �es�den��a� mac�op�age��, an���H��1 ��SA�111�� monoc�ona� an��bod�es and po�y�clonal anti-TGF-β1, anti-desmin, anti-collagen 1α, and, and and

an���f�b�onec��n an��bod�es �Se�o�ec ��d., �xfo�d, UK; S��essgene, V�c�o��a, B����s� Co�umb�a, Canada; C�em�con, Temecu�a, CA; San�a C�u�, CA�� we�e app��ed �n ���s s�udy fo� �mmuno��s�oc�em�s��y us�ng ��e s�anda�d �o�se�ad�s���e s�anda�d �o�se�ad�s�s�anda�d �o�se�ad�s� pe�ox�dase p�o�oco�. Se�um mac�op�age m�g�a��on �n��b���o�y fac�o� �MI��� �eve� was de�ec�ed by ��ISA acco�d�ng �o ��e �ns��uc��on of manufac�u�e� �C�em�con��.

Measurement of portal and systolic blood pressureAn�ma�s we�e anes��es��ed w��� �e�am�ne �100 mg/�g, �p��anes��es��ed w��� �e�am�ne �100 mg/�g, �p��w��� �e�am�ne �100 mg/�g, �p�� and Rompun �0.2 mg/�g, �p��. ��o��a� p�essu�e and sys�o��c b�ood p�essu�e we�e measu�ed by d��ec��y �n��oduc�ng 24� Ang�oca��® �BD B�osc�ences, San Jose, CA�� �n�o ��e po��a���e po��a�po��a� vein or abdominal aorta and connecting with a saline filleda saline filledsaline filled s��a�n gauge ��ansduce�. T�e s�gna�s we�e mon��o�ed by Co��n B���408 Ma�� III �Japan�� and we�e se� �o �e�o befo�e ��e measu�emen�.

HO enzymatic activityT�e H� en�yma��c ac��v��y was measu�ed by ��e p�oduc���e p�oduc�p�oduc���on of m�c�osoma� b����ub�n �n ��e ��ve�. ��o�en samp�es of ��ve�s we�e �omogen��ed �n �ce�co�d suc�ose and T���HC� buffe�. M�c�osoma� pe��e� was ob�a�ned af�e� cen���fu�M�c�osoma� pe��e� was ob�a�ned af�e� cen���fu��c�osoma� pe��e� was ob�a�ned af�e� cen���fu�ga��on and was ��en �e�suspended �n MgC�2�po�ass�umpo�ass�um p�osp�a�e buffe�. Samp�e p�o�e�n was fu���e� �ncuba�ed w��� ��e �eac��on m�x�u�e con�a�n�ng �a� ��ve� cy�oso�, �e�m�n, g�ucose�6�p�osp�a�e, g�ucose�6�p�osp�a�e de�yd�o�genase and NAD��H �S�gma�A�d��c�, S�. �ou�s, M��� fo� 60 m�nu�es a� 37 ℃. T�e gene�a�ed b����ub�n was measu�ed us�ng spec��op�o�ome��y. T�e �eve� of b����ub�n p�oduc���on was demons��a�ed by ��e �a��o of samp�e/no�ma� ��ve�.

RNase protection assayT�e mRNA �eve� of �a�ge� genes was de�e�m�ned bymRNA �eve� of �a�ge� genes was de�e�m�ned by RNase p�o�ec��on assay acco�d�ng �o ��e �ns��uc��on of manufac�u�e� �R�boQuan� ���, BD B�osc�ences ���a�m�n�gen, San D�ego, CA��. In b��ef, �o�a� RNA of ��ansduced and non���ansduced ��ve� samp�es we�e ex��ac�ed and pu���f�ed us�ng Rneasy ��� �Q�agen, H��den, �e�many��. T��ee m�c�og�ams of RNA/samp�es we�e �yb��d��ed w��� com�p��men�a�y [32��]UT�� �abe�ed ��bop�obes ove�n�g��. T�e p�obes we�e d�ges�ed w��� Rnase and we�e �oaded on a dena�u�ed po�yac�y�am�de ge�. T�e �ad�oac��ve s�gna�s we�e then detected by exposure to X-ray film (BioMax, Kodak, Rochester, NY) and quantified by phosphorimaging (Strom, Mo�ecu�a� Dynam�cs, Sunnyva�e, CA��.

Statistical analysisDa�a we�e demons��a�ed as mean ± S�M. �ne�way AN��VA was used �o compa�e ��e d�ffe�ence of means be�ween ��e expe��men�a� g�oups w��� ��e Bonfe��on�’s t��es�. P < 0.05 was considered statistically significant.

rESuLtS r���-mediated stable HO enzymatic activity in rat liver To en�ance H� ac��v��y �n a s�ab�e manne�, we adm�n��s�e�ed ��e �AAV ca��y�ng �a� H��1 cDNA �o ��e ��ve� ���oug� po��a� �njec��on. Af�e� ��e �njec��on, a �a�ge num�

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be� of H��1 pos���ve non�pa�enc�yma� ce��s we�e found �n bo�� �AAV/���� and �AAV/H��1���ansduced ��ve�s on d 7 �17.7 ± 0.5 ce��s/mm2 ve�sus 10.8 ± 0.7 ce��s/mm2, n = 3��. Howeve�, ��e numbe� of pos���ve ce��s dec�eased d�ama���ca��y �o a �eve� s�m��a� �o no�ma� ��ve�s �n �AAV/���� ��ansduced ��ve� on day 30, w�e�eas ��e�e was a s�gn�f��can��y �a�ge� numbe� of H��1�pos���ve non�pa�enc�yma� ce��s �n �AAV/H��1���ansduced ��ve�s �3.9 ± 0.8 ce��s/mm2 vs 5.8 ± 1.0 ce��s/mm2, n = 3, P < 0.05; ��gu�es 1A and 1B��. No H��1 pos���ve �epa�ocy�e was found �n bo�� �AAV/���� and �AAV/H��1 ��ea�men� g�oups on d 7. T�e ex�pression of HO-1 in hepatocyte was first detected sporadi�ca��y on d 14 af�e� po��a� �njec��on of �AAV/H��1, w�e�e�as ��e�e was no H��1 pos���ve �epa�ocy�e �n �AAV/�������ansduced ��ve�s �da�a no� s�own��. Mo�e H��1 pos���ve �epa�ocy�es cou�d be de�ec�ed �n �AAV/H��1���ansduced ��ve�s on d 30 �2.2 ± 0.3 ce��s/mm2, n = 3, P < 0.05��, w�e�eas ��ey �ema�ned unde�ec�ab�e �n �AAV/�������ansduced ��v�e�s ���gu�es 1A and 1B��. To eva�ua�e ��e b�o�og�ca� ac��v��y of ��ansduced H��1 �n ��e ��ve�s, we de�e�m�ned ��e en��yma��c ac��v��y of H� by measu��ng ��e gene�a�ed b����u�b�n of m�c�osoma� p�o�e�n �so�a�ed f�om ��e ��ve�s. T�e�e was a s�gn�f�can� �nc�ease of H� ac��v��y of ��e �AAV���ansduced ��ve�s on d 7 ���e ea���es� ��me po�n� ��a� we de�ec�ed��. Bo�� �AAV/���� and �AAV/H��1 ��ansduced ��ve�s s�owed �nc�eased amoun� of gene�a�ed b����ub�n �n �so�a�ed m�c�osoma� p�o�e�ns. Imp�ess�ve�y, ��e amoun� of gene�a�ed b����ub�n �0.9 fo�d ove� ��e basa� �eve� of no�ma� �a� ��ve��� �ema�ned e�eva�ed �n ��e �AAV/H��1 ��ea�men� g�oup and sus�a�ned fo� ove� 3 mo of obse�va��on ��me. In con��as�, ��e amoun� of gene�a�ed b����ub�n dec�eased �o ��e �eve� of no�ma� �a�s �n ��e �AAV/���� ��ea�men� group on d 14. (Figure 1C). This suggests the specificity of �AAV/H��1 �n ��e �nduc��on of s�ab�e H� ac��v��y �n �a� ��ve�.

Enhancement of HO activity suppressed the development of cirrhosis in rats In��agas���ca��y adm�n�s��a��on of CC�4 �o adu�� ���� �a�s fo� 9 w� �esu��ed �n ��e fo�ma��on of m�c�onodu�a� c�����os�s. To fu���e� eva�ua�e ��e effec�s of e�eva�ed H� ac��v��y on ��e deve�opmen� of c����os�s, we exam�ned va��ous pa�ame�e�s �e�a��ng �o c����os�s af�e� ove��exp�es�s�on of H��1 �n �a� ��ve� by �AAV gene ��ansfe� ���gu�e 2A��. H�s�o�og�ca� exam�na��on s�owed mass�ve amoun� of fibrotic tissues accumulated in the portal tract areas of non���ansduced o� �AAV/�������ansduced ��ve�s and �ed �o ��e fo�ma��on of m�c�onodu�a� c����os�s accompan�ed w��� po��a� �ype��ens�on and �owe� sys�o��c b�ood p�es�su�e �n ��e majo���y of �a�s. �AAV/H��1 gene ��ansfe� markedly diminished the amount of accumulated fibrotic ��ssues as s�own �n Masson’s ���c��ome �MT�� s�a�n�ng and �mmuno��s�oc�em�s��y. No m�c�onodu�a� c����os�s was formed and only minimal fibrotic tissues could be found �n ��e po��a� ��ac� a�eas of �AAV/H��1���ansduced ��ve�s (Figure 2B). Analysis of fibrotic areas using the computer sof�wa�e s�owed ��a� ��e�e was s�gn�f�can��y �ess f�b�o��c e�emen� depos���on �n ��e �AAV/H��1���ansduced ��ve� (��.9% ± 0.9% by MT staining, 3.1% ± 2.6% by collagen 1α

Figure 1 A: The expression patterns of HO-1 in the livers after portal injection of rAAV/HO-1 in LEW rats. Red arrow indicated the HO-1-positive non-parenchymal cells; green arrow indicated the hepatocyte; B: The quantification of the HO-1 positive cells in the liver after administration of rAAV/HO-1. Data were presented as number of HO-1 positive cells per mm2; n = 3-5, mean ± SE, aP<0.05; C: The HO enzymatic activity of rAAV/GFP or rAAV/HO-1 transduced livers. The data were demonstrated as the amount of generated bilirubin of microsomal protein from liver tissues and were shown as the fold changes over the normal rat liver, mean ± SE, n = 3, aP < 0.05.

0

0.5

1

1.5

2

2.5

1 2 4 12Time after portal injection (wk)

rAAV/GFP

rAAV/HO-1

aa

a

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2018 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol April 7, 2006 Volume 12 Number 13

rAAV/GFP rAAV/HO-1

Day

30

Day

7

0

1

2

3

Day 7 Day 30

rAAV/GFP

rAAV/HO-1

Non-parenchymal cell Hepatocyte

0

5

10

15

20

Day 7 Day 30

a

a

a

A

B

← ←

←←

←←

HO

-1 p

ositi

ve c

ells

/mm

2

HO

-1 p

ositi

ve c

ells

/mm

2

C

0

20

40

60

80

100

120

Normal

rat

Non-tr

ansd

uced

rAAV

/GFP

rAAV

/HO-1Sy

stol

icbl

ood

pres

sure

(mm

Hg)

0

2

4

6

8

10

12

14

16

Normal

rat

Non-tr

ansd

uced

rAAV

/GFP

rAAV

/HO-1

Port

alpr

essu

re(m

mH

g)

d

0

5

10

15

20

25

30

Non-transduced rAAV/GFP rAAV/HO-1

Fibr

oic

area

(%)

MT

Collagen 1

Fibronectin

b

bb

Figure 2 A: The model to induce micronodular cirrhosis in LEW rat. The rAAVs were injected through the portal vein shortly before the induction; B: The histology and immunohistochemistry of the livers with or without treatment at the 10th week (H-E, original magnification ×40; MT: Masson’s trichrome stiaining, the fibrotic elements were stained in green, original 100×; collagen 1 and fibronectin staining, original magnification ×100); C: The measurement of fibrotic area in the livers at the 10th week by the computer software as described in Materials & Methods. Data were shown as mean±SE, n = 5, bP < 0.001; D and E: The systolic and portal pressure of rats after induction of liver cirrhosis with or without treatments. The data were shown as mean±SE, n = 10, dP < 0.001.

s�a�n�ng, 4.4% ± 1.2% by f�b�onec��n s�a�n�ng, P < 0.001��. In con��as�, �n ��e ��ve�s of non���ea�men� o� �AAV/���� ��ea�ed g�oups, ��e�e was a ��g�e� �eve� of f�b�o��c e�e�men� depos���on �22.2% ± 5.8% o� 20.8 %± 3.8% by MT staining, 21.9 %± 5.��% or 24.5 %± 2.8% by collagen 1α s�a�n�ng, 24.1 %± 4.4% o� 21.1% ± 3.6% by f�b�onec��n staining, Figure 2C). In addition, the beneficial effects of H��1 on ��e deve�opmen� of ��ve� c����os�s cou�d be �e�flected by the reduction of portal hypertension (8.0 ± 0.8 mmHg ve�sus 13.3 ± 0.4 mmHg �n ��e non���ea�men� g�oup and 13.8 ± 0.6 mmHg �n ��e �AAV/���� ��ea�men� g�oup, P < 0.001), whereas there was no significant differ�ence a� ��e sys�o��c b�ood p�essu�e �81.7 ± 0.3 mmHg�� �n compa��son �o non���ea�men� �81.8 ± 0.6 mmHg�� o� vec�o� con��o�s �87.0 ± 0.2 mmHg, P > 0.31, ��gu�es 2D and 2���.

Stable HO activity protected against CCl4-mediated recur-rent liver injury

Repea�ed adm�n�s��a��on of CC�4 �o adu�� �a�s �ed �o c��on��c ��ve� �nju�y w��� e�eva�ed p�asma �eve� of �o�a� b����ub�n (no treatment group: 5��.25 ± 6.95 μmol/L; rAAV/GFP group: 60.5±4.8�� μmol/L) and ALT (non-treatment group: 4 522 ± 334 IU/�; �AAV/���� g�oup: 6 552 ± 1 363 IU/�, n = 5��. Inc�eas�ng H� ac��v��y �n ��e ��ve� by �AAV/H��1 s�gn�f�can��y �mp�oved ��e ��ve� func��on of �a�s unde� �ong��e�m CC�4 �ox�c��y, ��e amoun� of �o�a� b����ub�n and A�T dec�eased d�ama��ca��y �n ��e �AAV/H��1���ea�ed �a�s �n compa��son �o non���ea�men� o� �AAV/���� con�trols (total bilirubin: 6.20 ± 3.49 μmol/L; ALT: 199.6 ±80.5 IU/�, n = 5, P < 0.005, ��gu�es 3A and 3B��. To fu���e� exam�ne ��e p�o�ec��ve effec�s of ove��exp�ess�ng H��1 �n ��e ��ve�s, we de�e�m�ned ��e ��ansc��p� �eve� of apop�o��c genes and ene�gy exc�anges of ��ve�s unde� �ong��e�m CC�4 �ox�c��y. Rnase p�o�ec��on assay s�owed ��e ��ansc��p� level of Fas, caspase 3, and BAX genes significantly dimin��s�ed �n ��e �AAV/H��1���ansduced ��ve�s �n = 3, P < 0.05,

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Tsui TY et al . HO-1 suppresses liver fibrogenesis 2019

Non-transduced rAAV/GFP rAAV/HO-1

MT

Fibr

onec

tinCo

llage

n1

H&

E

rAAVs

Phenobarbital sodium + CCl4 (10 wk)A

B

0

2

4

6

8

10

12

14

16

Normal

rat

Non-tr

ansd

uced

rAAV

/GFP

rAAV

/HO-1

Port

alpr

essu

re(m

mH

g)

d

E

D

C

Figure 3 A and B: The liver function of rats with or with treatment under long-term CCl4 toxicity was presented by the determination of plasma ALT and total bilirubin level. The data was shown as mean±SEM, n = 5-10, aP<0.001; C and D: The representative picture of the expression patterns of apoptotic genes in the livers at the 10th week. The mRNA level of genes was detected by RNase protection assay. The quantification of the expression level was measured by phosphoimager as described in Materials & Methods. The data were shown as mean ± SE, n = 3-5, bP<0.05; E: The ATP level of the livers of rats with or without treatments at the 10th wk, n  = 5-10, dP<0.001.

��gu�es 3C and 3D��, w�e�eas ��ese genes we�e s��ong�y ex�p�essed �n ��e non���ansduced o� �AAV/�������ansduced ��ve�s. T�e benef�c�a� effec�s of H��1 on CC�4�med�a�ed c��on�c ��ve� �nju�y we�e a�so s�own by ��e �mp�ovemen� of ��ve� ene�gy exc�anges. �ve��exp�ess�on of H��1 by �AAV s�gn�f�can��y �mp�oved ��e �n��a�epa��c AT�� �eve�

Bcl-xL/S

Caspase 1

Caspase 3

BAX

L32

GAPDH

Fas

Caspase 2

Non- tr

ansd

uced

rAAV

/GFP

rAAV/H

O-1

0

2 000

4 000

6 000

8 000

10 000

Normal

rat

Non-tr

ansd

uced

rAAV/GFP

rAA

V/HO-1

ALT

(IU

/L)

a

0

20

40

60

80

Normal

rat

Non-tr

ansd

uced

rAAV

/GFP

rAAV

/HO-1

Tota

lbili

rubi

n(u

mol

/L)

a

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2020 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol April 7, 2006 Volume 12 Number 13

A

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Fas Bcl-xL/S Caspase 3 BAX

Intr

ahep

atic

mR

NA

leve

l(t

arge

tge

ne/G

APD

H)

Non-transduced

rAAV/GFP

rAAV/HO-1

bb

b

0

5

10

15

20

25

Normal

rat

Non-tr

ansd

uced

rAAV

/GFP

rAAV

/HO-1

Intr

ahep

atic

ATP

leve

l

(arb

itrar

yun

it)

d

E

D

C

B

�n �AAV/H��1��ansduced ��ve� �n compa��son w��� non���ea�men� o� �AAV/�������ansduced ��ve�s of �a�s unde� �ong��e�m CC�4 �ox�c��y �n = 3�5, P   < 0.05; ��gu�e 3���.

Stable HO activity suppresses the pro-inflammatory and pro-fibrogenic responses in CCl4-treated ratsTo characterize the effects of HO-1 on the chronic inflam�ma�o�y �esponse fo��owed by ��ve� �nju�y, we exam�ned ��e ��s�opa��o�og�ca� pa�ame�e�s of ��ve� ��ssues and ��ansc��p� o� p�o�e�n �eve� of p�o��nf�amma�o�y cy�o��ne�MI�. A �a�ge amoun� of �D1�pos���ve ��nf����a��ng�� mac�op�ages accumu�a�ed �n ��e po��a� ��ac� a�eas �n ��e non���ansduced o� �AAV/�������ansduced ��ve�s, w�e�eas on�y a few �D1�pos���ve mac�op�ages we�e found �n �AAV/H��1���ansduced ��ve�s ���gu�e 4A��. In con��as�, ��e�e was no significant change in the number of ED2-positive (tissue-�es�den��a��� mac�op�ages �n ��e ��ve�s of CC�4���ea�ed �a�s. In cons�s�en� w��� ��e �mmuno��s�oc�em�ca� s�a�n�ngs, de�ec��on of �n��a�epa��c mRNA �eve� by Rnase p�o�ec��on assay and se�um �eve�s of MI� by ��ISA me��od s�owed s�gn�f�can� �educ��on of ��e exp�ess�on �eve� of MI� �n �AAV/H��1���ea�ed �a�s �n compa��son �o non���ea�men� o� �AAV/���� con��o�s �n = 3�5, P < 0.001, ��gu�es 4B and 4C��.

To exam�ne ��e effec�s of �AAV/H��1 on ��e f��b�ogen�c p�ocess af�e� ��ve� �nju�y, we nex� exam�ned ��e

Figure 4 A: The representative pictures of pro-inflammatory and pro-fibrogenic responses in the livers of rats with or without treatment at the 10th week. Infiltrating macrophage (ED1), tissue residential macrophages (ED2), TGF-β1, and activated hepatic stellate cells (desmin) were detected by immunohistochemistry; original magnification, ×100; B: The profile of pro-inflammatory and profibrogenic cytokine expression. The level of mRNA was detected by RNase protection assay and was quantified by phosphoimager, n = 3-5, aP<0.05; C: The plasma level of macrophage migration inhibitory factor (MIF) was detected by ELISA. The data were shown as fold changes of over the level in normal rat, n = 3-5, bP < 0.001.

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Tsui TY et al . HO-1 suppresses liver fibrogenesis 2021

expression patterns of pro-fibrogenic cytokine-TGF-β1, w��c� p�ays an �mpo��an� �o�e �n ��e ac��va��on and ��ans��tion of HSCs to myofibroblast-like cells after liver in�ury.s to myofibroblast-like cells after liver in�ury. to myofibroblast-like cells after liver in�ury. Immunohistochemistry showed that TGF-β1 was highly exp�essed �n ��e po��a� ��ac� a�eas �n ��e ��ve�s of �ong��e�m CC�4���ea�ed �a�s. In con��as�, on�y a m�n�ma� numbe� of cells expressed TGF-β1 in rAAV/HO-1-transduced ��ve�s of �a�s unde� ��e same CC�4 p�o�oco� ���gu�e 4A��. ��a�a��e� �o ��e �mmuno��s�oc�em�ca� s�a�n�ngs, RNase p�o�tection assay also showed significant reduction in the tran�script level of TGF-β1 in rAAV/HO-1-transduced livers �n = 3�5, P < 0.001, Figure 4B). This finding was associated w��� ��e dec�eased numbe� of ac��va�ed HSCs �desm�n�pos���ve�� �n �AAV/H��1���ansduced ��ve�s ���gu�e 4A��.

dIScuSSIonT�e�e �s a w�de �ange of fac�o�s can �esu�� �n �epa�oce���u�a� �nju�y. Howeve�, ��e p��ma�y �esponse �ead�ng �o sub�sequen� p�o��nf�amma�o�y and p�o�f�b�ogen�c �esponses �n ��ve� �s ex��ao�d�na�y s�m��a� [14]. Ana�ys�s of c��n�ca� pa�ame�e�s based on ��e ��ea�men� of p��ma�y ��ve� d�s�ease s�owed encou�ag�ng �esu��s �n ��e dec�ease of ��e severity of liver fibrosis/cirrhosis [15,16]. Howeve�, none of anti-fibrotic treatment has been shown clinically effective. A numbe� of ��e�apeu��c app�oac�es �a�ge��ng on an���oxidant, anti-inflammatory response, suppression of HSC ac��va��on, �nduc��on of HSC apop�os�s o� �nc�ease of ��e deg�ada��on of ex��ace��u�a� ma���x �ave been s�own effec�tive in the attenuation of the severity of liver fibrosis/cir���os�s �n va��ous an�ma� mode�s [14]. Mos� of ��em, �owev�e�, canno� eas��y ��ans�a�e �o ��e c��n�ca� se���ng, because ��e majo���y of app�oac�es focus on ��e s�ng�e s�ep of d�sease p�og�ess�on and ��e c��n�ca� s��ua��on �s fa� mo�e comp�ex ��an ��a� �n ��e expe��men�a� se���ng. T�us, �� �s s���� u�ged to find out a therapeutic target that has multiple effects on d�sease p�og�ess�on and can be app��ed po�en��a��y �n ��e c��n�ca� se���ng.

HO-1-mediated anti-apoptotic and anti-inflammatory ac��v���es a�e among ��e mos� a���ac��ve mec�an�sms �n ce��u�a� p�o�ec��on [17]. In fac�, H��1 on�y exp�esses �n a subpopu�a��on of Kupffe� ce��s �n no�ma� ��ve�, w�e�eas a sma�� amoun� of H��2 �s cons���u��ve�y be�ng exp�essed �n �epa�ocy�e [3,4]. None of ��em exp�esses �n s�nuso�da� endo��e��a� ce��s and �epa��c s�e��a�e ce��s [3]. Howeve�, a significant increase in HO-1 expression was observed on ��e w�o�e o�gan �eve� unde� s��ess cond���ons [4]. In ��e ��ve� w��� acu�e �nju�y, H��1 was exp�essed �n ��e majo���y of Kupffer cells and infiltrating macrophages, which might func��on as a feedbac� �oop �o con��o� ��e mac�op�age ac��va��on [18�20]. A���oug� ��e w�o�e H� ac��v��y �nc�eases d�ama��ca��y, ��e need fo� ��e ��ve� �o ove�come ��e �nsu��s of ce��u�a� �nju�y seems �o be �e�a��ve�y �nsuff�c�en�. In�c�eas�ng H� ac��v��y by �n��oduc��on of exogenous H��1 by rAAV/HO-1 in our model that significantly improved ��e �ong��e�m ou�comes of CC�4��nduced ��ve� c����os�s cou�d be based on �1�� ��e an���apop�o��c effec�s of H��1 on hepatocyte; (2) the anti-inflammatory effects of HO-1 on ��e con��o� of ��e ce��u�a� �esponse of �epa�ocy�e �n ��e p�oduc��on of MI� unde� s��ess and/o� �nju�y cond��

A

Non-transduced rAAV/GFP rAAV/HO-1ED

2D

esm

inTG

F-β

1ED

1

TGF β1TGF β3

Lt β

MIF

L32

GAPDH

Non-tr

ansd

uced

rAAV/G

FP

rAAV/H

O-10.0

0.2

0.4

0.6

0.8

1.0

MIF TGF-β1

Intr

ahep

atic

mR

NA

leve

l(t

arge

tge

ne/G

APD

H)

Non-transduced

rAAV/GFP

rAAV/HO-1

a

a

0

0.5

1

1.5

2

2.5

Normal

rat

Non-tr

ansd

uced

rAAV

/GFP

rAA V /H

O-1Fold

chan

ges

ofpl

asm

aM

IFle

vel

b

C

B

��ons and ��e supp�ess�on of ��e mac�op�age ac��va��on; and �3�� ��e an���f�b�ogen�c effec�s of H��1 on ��e sup�p�ess�on of ��e co��agen syn��es�s and/o� p�o��fe�a��on of ac��va�ed HSCs.

Imp�ovemen� of ��ve� func��on unde� �ong��e�m CC�4 �ox�c��y by �AAV/H��1 may �ef�ec� ��e fac� ��a� ��e ex�p�ess�on of H��1 �n ��e �epa�ocy�e was ab�e �o p�even� ��ve� damage, w��c� was suppo��ed by ��e down��egu�a��on of p�o�apop�o��c genes and en�ancemen� of ��ve� AT�� �eve� �n ou� mode�. In acu�e ��ve� �nju�y and �sc�em�a/�epe�fus�on �nju�y of ��ansp�an�ed ��ve�, �ecen� da�a sug�ges�ed ��a� ��e �es�s�ance of H��1 exp�ess�ng ce��s �o ��e p�o�apop�o��c s��mu�� m�g�� be d��ec��y ���oug� ��s en�y�ma��c p�oduc� C� and/o� �nd��ec��y ���oug� ��e �nduc��on of ��e �e2+�seques�e��ng p�o�e�n fe�����n. �xposu�e of C� �o ��e p��ma�y �epa�ocy�e cou�d p�even� ��e �umo� nec�o�sis factor-α-mediated and anti-CD95-mediated apoptotic even�s ���oug� ��e down��egu�a��on of caspase�3 ac��v���y [21], w�e�eas ��e �nduc��on of fe�����n supp�essed se�um�dep��ved o� ox�da��ve s��ess�med�a�ed �epa�ocy�e apop�os�s by modu�a��on of �n��ace��u�a� �e2+ �eve� and �n��b���on of �e2+�med�a�ed conve�s�on of �yd�ogen pe�ox�de �n�o �H− and �H ���oug� ��e �en�on �eac��on [7,22]. In add���on �o an���apop�o��c effec�s, H��1 cou�d a�so supp�ess ��e p�oduc���on of p�o��nf�amma�o�y cy�o��ne�MI� p�oduc��on �n a �uman p��ma�y �epa�ocy�e cu��u�e �Tsu� TY, e� a�. unpub���s�ed da�a��. T��s may sugges� ��a� H��1 can func��on as �omeos�a��c mo�ecu�e �n ��e p�even��on of apop�o��c even� and ��e con��o� of ce��u�a� �esponse.

H��1 can be �nduced �n va��ous ce�� �ypes �nc�ud�ng HSC ���oug� d��ec� o� �nd��ec� mec�an�sms. �u� da�a a�e cons�s�en� w��� a �ecen� f�nd�ng �n p��ma�y �uman HSC cu��u�e s�ow�ng ��a� ��e �nduc��on of H��1 exp�ess�on �n HSC suppressed the transcript level of pro-collagen 1α and se�um�med�a�ed HSC p�o��fe�a��on [23]. T��s may sugges� ��a� H��1 can se�ve as a nega��ve �egu�a�o� �n ��e con��o� of HSC ac��va��on and p�o��fe�a��on. T�us, �ess accumu�a�tion of fibrotic elements and prevention of the develop�men� of po��a� �ype��ens�on �n �AAV/H��1���ansduced animals might reflect the outcomes of cellular protection, anti-inflammatory and anti-fibrogenic responses. Although fu���e� �nves��ga��on �s needed �o c�a��fy ��e mec�an�sms �n mo�e de�a��s, �nc�eased H� ac��v��y �n ��ve� and sys�em�c effec�s of ��e p�oduc�s of H� en�yma��c ac��v��y can be ��e �ey �n ��e con��o� of ��e deve�opmen� of c����os�s and po��a� �ype��ens�on �n ou� mode�.

In conc�us�on, ou� da�a demons��a�ed ��a� en�ance�men� ��e H� ac��v��y �n a s�ab�e manne� can supp�esses ��e pa��op�ys�o�og�ca� c�anges of c����os�s. In add���on, �AAV�med�a�ed gene ��ansfe� may �ep�esen� an a���ac��ve app�oac� �n con��o���ng ��e deve�opmen� of c����os�s. Howeve�, fu���e� s�ud�es s�ou�d be ca���ed ou� �n o�de� �o answe� ��e ques��on of w�e��e� ��e �AAV app�oac� �s su��ab�e fo� ��e �epa����s v��us�med�a�ed c��on�c ��ve� �nju�y and ��e subsequen� ou�come.

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