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GASTROENTEROLOGY 2004;126:1122–1133

ASIC–LIVER, PANCREAS, AND BILIARY TRACT

reatment With Human Metalloproteinase-8 Gene Deliverymeliorates Experimental Rat Liver Cirrhosis

ERNANDO SILLER–LOPEZ,* ANA SANDOVAL,* SILVIA SALGADO,* ADRIANA SALAZAR,*IRIAM BUENO,* JESUS GARCIA,* JOSE VERA,* JAVIER GALVEZ,* IVAN HERNANDEZ,*ARTHA RAMOS,* ESTUARDO AGUILAR–CORDOVA,‡ and JUAN ARMENDARIZ–BORUNDA*,§

Institute for Molecular Biology in Medicine and Gene Therapy, Centro Universitario de Ciencias de la Salud, University of Guadalajara,uadalajara, Mexico; ‡Advantagene, Inc., San Diego, California; and §Organismo Publico Descentralizado Hospital Civil Guadalajara,

uadalajara, Mexico

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ackground & Aims: An extrahepatic human neutrophilollagenase complementary DNA (matrix metallopro-ease-8) cloned in an adenovirus vector was used as aherapeutic agent in cirrhosis. Methods: A high titer oflinical-grade AdMMP8 was obtained. Results: HeLaells transduced with AdMMP8 expressed recombinantatrix metalloprotease-8 messenger RNA and matrixetalloprotease-8 protein. Matrix metalloprotease-8 inulture sups showed enzymatic activity against nativeollagen type I, which was inhibited by ethylenedia-inetetraacetic acid, 1,10-phenanthroline, and tissuenhibitor of metalloprotease-1. In vivo transductionhowed matrix metalloprotease-8 activity, and studieso establish the efficacy of this characterized vectorere performed in CCl4 and bile duct–ligated cirrhoticats. Transduction with 3 � 1011 viral particles per kilo-ram resulted in hepatic detection of both messengerNA and protein matrix metalloprotease-8. A consistentesponse in fibrosis reversal was observed in CCl4 rats.iver fibrosis in bile duct–ligated cirrhotic animals wasecreased in 45%, along with diminished hydroxyprolineontent, after AdMMP8 treatment. The expression ofatrix metalloprotease-2 and matrix metalloprotease-3as up-regulated in AdMMP8 rats. Free tissue inhibitorf metalloprotease-1, as an indirect measurement ofctive uncomplexed matrix metalloproteases, waslso increased in the AdMMP8 groups. Transformingrowth factor-� messenger RNA was diminished, andatrix metalloprotease-9 and hepatocyte growth fac-or increased. Treatment in both models correlatedith improvements in ascites, functional hepaticests, and gastric varices, indicating diminished intra-epatic blood pressure in animals injected withdMMP8. Conclusions: Therefore, therapy with theatrix metalloprotease-8 gene is promising for use inclinical setting.

ur research has been focused on the cellular andmolecular mechanisms that govern the progression

nd potential reversion of fibrotic processes in both ex-erimental and human cirrhosis. Liver cirrhosis is anrreversible end-stage disease and is the seventh leadingause of mortality worldwide. Generally, chronic con-umption of alcohol is the main cause of cirrhosis, al-hough chronic infection with viral hepatitis C is also aajor cause.1 Normally, the homeostatic hepatic cell/

xtracellular matrix (ECM) ratio is maintained in theiver by well-balanced synthesis and degradation of ECMomponents.2 In cirrhotic liver, however, an altered bal-nce takes place because of an excess in the synthesis andeposition of ECM proteins (fibrogenesis) and/or a reduc-ion in the removal of this excess (fibrolysis), with theonsequent onset of fibrotic scarring. The pathophysiol-gy of ECM formation during hepatic fibrosis is multi-aceted and complex. Fibrogenesis involves a change inhe expression of ECM proteases (matrix metallopro-eases; MMPs) or their inactivation by specific inhibitorstissue inhibitors of metalloproteases; TIMPs)3,4 and anncrease in the synthesis of interstitial (types I and III)nd basement membrane (type IV) collagens, fibronectin,nd proteoglycans driven by signaling pathways medi-ted by proinflammatory cytokines such as transformingrowth factor (TGF)-�, interleukin-1, and platelet-de-ived growth factor. These cytokines are producedainly by activated Kupffer cells, which in turn activate

Abbreviations used in this paper: BDL, bile duct ligation; bp, baseair; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbentssay; HGF, hepatocyte growth factor; HSC, hepatic stellate cell; MMP,atrix metalloprotease; PCR, polymerase chain reaction; PCNA, pro-iferating cell nuclear antigen; TGF, transforming growth factor; TIMP,issue inhibitor of metalloprotease; VP, viral particle.

© 2004 by the American Gastroenterological Association0016-5085/04/$30.00

doi:10.1053/j.gastro.2003.12.045

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uiescent hepatic stellate cells (HSC), giving them aajor role in scarring organ tissue.2,5–7

For this reason, conventional therapies seeking a cureor hepatic cirrhosis have mainly focused on preventingr reducing the biosynthesis of collagen and/or increasinghe synthesis or activity of MMPs responsible for ECMegradation.5,8 With the advent of gene therapy, novelpproaches have been used to heal liver cirrhosis.9,10 Ourrevious work with urokinase-plasminogen activatorene delivery has shown promise. Human urokinase-lasminogen activator treatment of cirrhotic rats resultedn reversion of liver fibrosis and in vigorous hepatic cellegeneration.11

This work focuses on restoration of normal liver ar-hitecture with consequent recovery of liver function ofirrhotic animals by adenoviral administration of a geneoding for MMP-8, a neutrophil collagenase whose pref-rential substrate is type I collagen.12,13 The rationale forsing MMP-8 consisted of promoting in situ degradationf ECM proteins, releasing hepatic growth factors, andreeing up space for hepatic cell proliferation. We con-tructed an adenovirus vector (AdMMP8) that has provenfficient transduction and induced MMP-8 productionith properties of the naturally occurring protein, whichegraded efficiently in vitro in collagen type I and wasnhibited by ethylenediaminetetraacetic acid (EDTA),IMP-1, and 1,10-phenanthroline. Furthermore, becausee had shown that cirrhotic liver11,14 transfection by

denovirus vectors was dose dependent and that dosingn the range of 1011 viral particles (VP) per kilogram wasost effective in inducing a therapeutic effect, we de-

ided to use a single application of 3 � 1011 VP perilogram of AdMMP8 via the iliac vein in severelyirrhotic rats. This resulted in in situ production of theognate protein effective against collagen type I and inmelioration of the cirrhotic process. We used 2 differentxperimental cirrhosis models to validate our proof ofoncept, and we provided mechanistic insights on theolecular action of MMP-8.

Materials and MethodsRecombinant Plasmid AdenoviralConstruction

pAdHM2-MMP-8, an �E1 �E3 adenoviral plasmidector expressing the proenzyme of human MMP-8 under theontrol of the cytomegalovirus promoter (pCMV) and with aimian virus 40 polyadenylation signal, was constructed by ann vitro ligation method according to the method describedelow.15 The expression cassette coding for MMP-8 was ex-ised with NruI/ScaI from plasmid pcDNA-MMP-8 and in-erted into the SmaI site of the multiple cloning site of thehuttle plasmid pHM3, and the resulting plasmid, pHM3-

MP-8, was digested with I-CeuI and PI-SceI. The releasedragment was ligated with the I-CeuI/PI-SceI–digested plasmidAdHM2, which contains the �E1–�E3 adenovirus type 5enome. The resulting ligation product was transformed intoltracompetent DH5a, and recombinant clones were isolatedy ampicillin selection and screened by restriction analysis.arge-scale preparation of recombinant plasmid pAdHM2-MP-8 was performed after positive identification.

Generation of Adenoviral Vector

AdMMP8, a replication-defective adenoviral vector,as obtained from pAdHM2-MMP8 by PacI digestion and

ransfected with lipofectamine into human kidney embryonic93 cells seeded in a 6-well plate by following the manufac-urer’s instructions. Twenty-four hours later, cells were cul-ured in minimal essential medium/5% BCS or overlaid with.5% agarose/minimal essential medium/10% BCS. Ten daysfter transfection, plaques showing a cytopathic effect weresolated, and viruses were amplified by transduction of humanidney embryonic 293 cells seeded in a 100-mm dish. Recom-inant viruses were harvested and plaque-purified twice andxpanded in 293 cells for preparation of large-scale stocks.olymerase chain reaction (PCR) was used for identification ofdMMP8 in every step of purification and for the final pro-uction. Vector production (including AdGFP as an irrelevantontrol vector) was performed under Good Laboratory Practiceonditions by using standard methods as previously de-cribed.16 Stocks were stored at �70°C and thawed immedi-tely before use. Recombinant adenovirus was titered on 293ells by end-point dilutions16 and tested for replication defi-iency by titration on nonpermissive HeLa cells.

Matrix Metalloprotease-8 Expression inHeLa Cells Infected With AdMMP8

Confluent HeLa cells obtained 24 hours after seedingn a 100-mm dish were incubated with AdMMP8 1 � 1010

P per milliliter (2.7 � 109 IU/mL) in minimal essentialedium/5% BCS, and medium was replaced with X-Vivo15

erum-free medium 24 hours later. Cellular lysate and super-atant serum-free medium were re-collected at 48 hours afterransduction for successive analysis. Serum-free media super-atant was treated with 1 mmol/L phenylmethylsulfonyl flu-ride, and 0.05% Brij35 and subsequently concentrated 5-foldn a Centricon-50. Protein content was determined by theradford method17 and stored at �20°C until assay.

Gene Expression by Reverse-TranscriptionPolymerase Chain Reaction

A total of 2 �g of total RNA was incubated with 1 Uf ribonuclease-free deoxyribonuclease I at 37°C for 30 min-tes, and the reaction was terminated by the addition of 50mol/L EDTA and heating at 75°C for 10 minutes. Samplesere incubated with 0.5 mg of oligo(dT)18 primer at 70°C for0 minutes and subjected to 100 U of Moloney murineeukemia virus reverse transcriptase (RT) for 50 minutes at

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1124 SILLER-LOPEZ ET AL. GASTROENTEROLOGY Vol. 126, No. 4

7°C; the same samples without RT were used as control. PCRmplification of the MMP-8 complementary DNA (cDNA)ragment corresponding to nucleotides 439–797 (358 baseairs [bp]) of the published human sequence (GenBank acces-ion no. J05556) was performed as previously described18 withaq polymerase for 30 cycles (94°C for 1 minute, 56°C for 1inute, and 72°C for 1 minute) by using sense primer com-

lementary to base pairs 439–462 (5�-AGCTGTCAGAG-CTGAGGTAGAAA-3�) and antisense primer complemen-

ary to base pairs 774–797 (5�-CCTGAAAGCATAGTTG-GATACAT-3�). PCR for the constitutively expressed humanousekeeping gene glyceraldehyde-3-phosphate dehydroge-ase (GenBank accession no. X01677) was performed with Taqolymerase for 30 cycles (94°C for 1 minute, 69°C for 1inute, and 72°C for 1 minute) by using sense primers

omplementary to base pairs 396–414 (5�-GCAGGGGG-AGCCAAAAGGG-3�) and antisense primer complementary

o base pairs 942–961 (5�-TGCCAGCCCCAGCGTCAAAG-�). The size of the expected product was 565 bp.19

Likewise, total RNA was extracted from livers of controlnd experimental animals to conduct RT-PCR for hypoxan-hinephospho-ribosyltransferase and hepatocyte growth factorHGF); collagen type I, III, and IV; TGF-�1; and MMP-9 tonalyze hepatic regeneration and mechanisms induced by ex-ression of MMP-8.

In Vitro and In Vivo Quantification of MatrixMetalloprotease-8 Secretion by Enzyme-Linked Immunosorbent Assay

Secretion of MMP-8 into the media supernatants andell lysates from uninfected and infected cells was quantifiedy using a Biotrak enzyme-linked immunosorbent assayELISA) according to the manufacturer’s instructions. Dilu-ions of the samples were effectuated to work within thetandard range of the assay. For in vivo determination, liverxtracts were obtained essentially as described previously11 andssayed with the same human ELISA as described previously.

In Vitro and In Vivo Collagenase ActivityAgainst Soluble Type I Collagen

Type I collagen proteolytic activity was analyzed asescribed elsewhere.12,20 To activate the MMP-8 proenzyme,liquots (10 �L) of the concentrated media supernatants werencubated for 4 hours at 25°C with 1 mmol/L APMA in auffer containing 50 mmol/L Tris (pH 7.5), 0.2 mol/L NaCl,0 mmol/L CaCl2 , 1 mmol/L ZnCl2 , 0.05% Brij35, and 100mol/L arginine. Pretreated samples were then incubated for

6 hours at 27°C with 10 �g of soluble calf skin type Iollagen in the absence or presence of the MMP inhibitors,10-phenanthroline (2 mmol/L), EDTA (20 mmol/L), orIMP-1 (200 nmol/L) in a final volume of 50 mL. For in vivonalysis, MMP-8 proteolytic activity was assessed in liveromogenates. Proteins were extracted with 6 mol/L of guani-ine HCl for 3 days. Proteins were then dialyzed againsthosphate-buffered saline for 2 hours, and sodium dodecylulfate was added to a final 1%. Protein concentrations were

etermined by the Bradford method.17 A total of 500 �g ofotal protein was incubated for 4 hours at 25°C with sameuffer as described previously and was then incubated for 16ours under the same conditions.The reaction products were analyzed on an 8% sodium

odecyl sulfate-polyacrylamide gel. The gel was stained withoomassie Brilliant Blue R-250 and destained with 30%ethanol and 10% acetic acid.

Animal Models of Cirrhosis

Basically, the regimen for CCl4 intoxication was per-ormed during 8 weeks, as described previously,11 and allnimal studies were performed on male Wistar rats in accor-ance with University of Guadalajara’s animal experimenta-ion guidelines. This experimental animal model closely re-embles human alcohol-induced cirrhosis. Rats weighing50–200 g were anesthetized with ethyl ether, and the com-on bile duct was exposed and ligated to induce fibrosis by

otal biliary obstruction (bile duct ligation; BDL) according tohe method of Lee et al.21 After surgery, rats were kept for 4eeks and were given free access to food and water throughout

he experiment. Five rats were sham-operated at the same timend used as controls. For the experiments aimed to show prooff concept on fibrosis reversion, at least 5 rats were used forach experiment. That is, 10 rats were ligated as describedreviously for 4 weeks, and a liver biopsy sample was obtainedrom each animal before and after the surgical procedure.hen, a biliodigestive anastomosis was established in eachnimal to re-establish bile flow to eliminate the injuriousbrogenic cause. Animals were left at this stage for 7 days, andliver biopsy sample was also taken. At this point, 5 animals

eceived 3 � 1011 VP per kilogram of either AdMMP8 ordGFP and 10 days later were killed. The fourth and final

iver biopsy sample was obtained for each animal. The fibrosisndex in multiple liver biopsy samples from animals at differ-nt stages was then determined.

Quantitative Analyses of Fibrotic Indexand Immunohistochemistry

Essentially, fibrotic index and immunohistochemistryere analyzed according to our previous reports, in whichultiple 5-mm-thick liver sections from AdMMP8, AdGFP,

nd saline-treated rats were stained with Masson’s dye and inhich morphometric image analyses (20 different microscopicelds in each tissue section) were performed by a computer-ssisted Image program (Qwin Leica).11,22,23 Histological find-ngs, cell necrosis, and hepatic rearrangement after treatmentere confirmed independently by 2 pathologists blinded to the

tudy.Hepatic cell proliferation was determined with standard

echniques by using a monoclonal anti–proliferating cell nu-lear antigen (PCNA) antibody (Boehringer Mannheim,annheim, Germany) at a 1:20 dilution in phosphate-buffered

aline and by registering the number of positive cells asescribed previously by using a computerized image program

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April 2004 CIRRHOSIS IMPROVEMENT BY MMP–8 GENE TRANSFER 1125

nd checking 20 random microscopic fields for each tissueection.

Biochemical Determinations ofHydroxyproline Content

Liver samples were obtained at the moment of death,nd 150 mg was subjected to acid hydrolysis to determine themount of hydroxyproline according to Rojkind and Gonza-ez.24

Western Blot Analysis

The presence of MMP-2, MMP-3, and TIMP-1 in liveromogenates from treated rats was shown by Western blotnalysis, as described previously.25 Approximately 100 mg ofontrol and experimental liver tissue was homogenized in lysisuffer (10 mmol/L HEPES [pH 7.9], 0.42 mol/L NaCl, 1.5mol/L of MgCl2 , 0.5 mmol/L dithiothreitol, 0.5% Nonidet-40, and 25% glycerol) with a protease inhibitor cocktail

rom Roche (Summerville, NJ) at 4°C, followed by centrifu-ation for 30 minutes at 13,000 rpm. Supernatant was col-ected, and protein concentration was determined by the Brad-ord assay.17 A total of 20 �g of protein for MMP-2 and

MP-3 and 300 �g for TIMP-1 was separated by electro-horesis on 10% acrylamide/sodium dodecyl sulfate gels andransferred to polyvinylidene difluoride membranes. Blotsere blocked in 5% nonfat dry milk in Tris-buffered saline for.5 hours; incubated for 1.5 hours in primary antibody diluted:1000 for MMP-2, 1:5000 for MMP-3, and 1:500 for TIMPpolyclonal antibodies; Santa Cruz Biochemicals, Santa Cruz,A) in 0.5% blocking buffer; and then incubated for 1 hourith horseradish peroxidase– conjugated polyclonal secondary

ntibody (from Sigma [St. Louis, MO] for the MMPs and fromoche for TIMP-1) diluted 1:5000 for MMP-2 and MMP-3

nd 1:3000 for TIMP-1. Proteins were detected with enhancedhemiluminescence detection reagents.

Statistical Analysis

Results are expressed as mean � SD. The Student t testas used. P � 0.05 was considered to indicate a significantifference between groups.

ResultsThe construction strategy for AdMMP8 cDNA

onsisted of cloning the MMP-8 expression cassette intohe pHM3 shuttle plasmid, and the recombinant adeno-iral plasmid pAdHM2-MMP-8 was generated by diges-ion of pHM3-MMP-8 and pAdHM2 with endonucle-ses I-CeuI and PI-SceI, as described by Mizuguchi anday.15 The recombinant adenoviral vector AdMMP8 wasenerated after transfection of pAdHM2-MMP-8 intoubconfluent 293 cells, and plaques from the first andecond round of amplification and from the final charac-erized production were identified by the appearance ofytopathic effects and by PCR for the MMP-8 gene (dataot shown).

Matrix Metalloprotease-8 Messenger RNAExpression in AdMMP8-TransducedHeLa Cells

HeLa cells were transduced with 1 � 1010 VP perilliliter AdMMP8, and 48 hours later cells were har-

ested for the analysis of MMP-8 messenger RNAmRNA) expression by RT-PCR analysis by using a setf oligonucleotides specific for human neutrophil colla-enase (Figure 1A). MMP-8 PCR product was absent inontrol cells, but mRNA for MMP-8 was constantlyetected after AdMMP8 transduction. Expression of theousekeeping gene glyceraldehyde-3-phosphate dehy-rogenase in nontransduced and transduced HeLa cellsas constant and not affected by treatment (Table 1).

Expression of Matrix Metalloprotease-8Protein in AdMMP8-Transduced HeLa Cells

The levels of MMP-8 protein expression and se-retion in control and AdMMP8-transduced HeLa cells

igure 1. (A) PCR products of MMP-8 and glyceraldehyde-3-phos-hate dehydrogenase (GAPDH) of total RNA extracted from controllanes 1 and 2) and AdMMP8-transduced (lanes 3 and 4) HeLa cells,reated without (lanes 1 and 3) or with (lanes 2 and 4) Moloney murineeukemia virus reverse transcriptase. PCR was performed with spe-ific primers for human MMP-8 and GAPDH. A DNA molecular 1-kilo-ase DNA ladder (Promega, Madison, WI) was used as a size marker.B) Collagenolytic activity of culture supernatants from control anddMMP8-transduced HeLa cells. Soluble native type I collagen was

ncubated alone (lane 1) or with media supernatant from AdMMP8-ransduced HeLa cells pretreated without (lane 2) or with (lanes 3–7)mmol/L APMA. Collagen was incubated in the presence of EDTA 20mol/L (lane 4), 1,10-phenanthroline 2 mmol/L (lane 5), and TIMP-10 nmol/L (lane 6) or 200 nmol/L (lane 7). (C) MMP-8 enzymaticctivity in cirrhotic liver homogenates transduced with AdMMP8 3 �011 viral particles per kilogram (lane 1), administered with AdGFP

lane 2) and collagen type I (lane 3). Collagen dimers and monomersre depicted as and �, 1A, and 2A and are the characteristic 3/4nd 1/4 products of the degradation of collagen chains 1 and 2,espectively.

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1126 SILLER-LOPEZ ET AL. GASTROENTEROLOGY Vol. 126, No. 4

ere quantified by ELISA (Table 2) by using a polyclonalntibody against human neutrophil MMP-8 that doesot cross-react with MMP-1, -2, -3, -7, -9, or -13 orT1-MMP. Pro-MMP-8 and active MMP-8 (both free

nd TIMP complexed) can be detected by this ELISAystem. There was a 1300-fold increase in the secretion ofMP-8 to the extracellular media after the infection ofeLa cells by AdMMP8 as compared with uninfected

ells. Conversely, a 23-fold increase of intracellularMP-8 was induced by adenoviral transduction.Levels of MMP-8 in the filtrate (i.e., proteins �50

ilodaltons) after Centricon concentration of culture su-ernatants were negligible, and collagenolytic activityas not detected in this fraction (data not shown), whichenotes the presence of MMP-8 in culture supernatantsn the form of either latent (85 kilodaltons) or active64–67 kilodaltons) form.

Collagenolytic Matrix Metalloprotease-8Activity of Media Supernatant in AdMMP8-Transduced HeLa Cells

HeLa cells were transduced with AdMMP8 1 �010 VP per milliliter for 48 hours and 24-hour serum-ree media supernatant was harvested for analysis ofroteolytic activity against collagen type I.AdMMP8 codes for the gene of latent MMP-8, which

fter expression requires the proteolytic (e.g., serine pro-eases or trypsin) or chemical (e.g., APMA or HgCl2)emoval of the pro-domain to become functionally active.

able 1. Primer Sequences and PCR Conditions for Genes A

Gene Primer sense

PRT 5�-TCC CAG CGT CGT T TAG TG-3� 5�-GGC TTTGF 5�-GCC ATG AAT TTG ACC TCT ATG AA-3� 5�-TTT AAT TOL I 5�-CAA GAA TGG CGA CCG TGG TGA-3� 5�-CTA CCGOL III 5�-AGA TGG ATC AAG TGG ACA-3� 5�-CAT GTTGF-� 5�-GCC TCC GCA TCC CAC CTT TG-3� 5�-GCG GGTMP-9 5�-TTG AGT CCG GCA GAC AAT CC-3� 5�-CCT TAT

OL, collagen; HPRT, hypoxanthinephospho-ribosyltransferase.

able 2. MMP-8 Expression in Control and AdMMP8-Transduced HeLa Cells

Variable

Levels of MMP8 (ng MMP-8/mg protein)

CL/SSupernatant (S) Cellular lysate (CL)

ontrol 0.07 � 0.04 13.7 � 2.3 196dMMP8 97.0 � 8.1 317.2 � 14.9 3.3

OTE. MMP-8 protein of culture supernatant and cell lysate of HeLaells were measured by ELISA. Data represent the mean � SEM of 4eplicates.

onetheless, it can be spontaneously activated in vivo byechanisms not yet elucidated.26

Culture media from nontransduced HeLa cells showedhe same pattern of protein fragments as those fromdMMP8-transduced HeLa cells without the organo-ercurial APMA preincubation (Figure 1B; lanes 1 and

). APMA-preactivated supernatant efficiently degradedoluble native type I collagen into the characteristic 1A3/4) and 2A (1/4) cleavage products (lane 3). Thenown chemical inhibitors EDTA (lane 4) and 1,10-henanthroline (2 mmol/L; lane 5) strongly inhibitedMP-8 activity. The physiological inhibitor TIMP-1

howed a progressive decrease in the degradation of theubstrate at increasing concentrations of TIMP-1: 50mol/L (lane 6) or 200 nmol/L (lane 7). CollagenolyticMP-8 activity in the hepatic tissue of cirrhotic animals

ransduced with AdMMP8 is shown in Figure 1C.Homogenates from hepatic tissue were analyzed for

roteolytic activity against native collagen type I. Evenhough a pro–MMP-8– coding cDNA was administered,nternal mechanisms not yet elucidated seem to be actingo convert it into active MMP-8. Figure 1C clearly showsow collagen I is degraded when it is incubated withamples from liver homogenates transduced withdMMP8; this is not observed with the GFP-treated

nimals.

AdMMP8-Directed Matrix Metalloprotease-8Gene Expression in Cirrhotic Rat Livers

Hepatic cirrhosis is characterized by an alteredalance between synthesis and breakdown of ECM pro-eins, mostly collagen type I. To push the ECM degra-ation and free up space for hepatocyte cell expansion, weursued the AdMMP8-driven degradation of exacerbatedollagen deposited in animals with cirrhosis induced forweeks with CCl4.We tested several doses and found that a single intra-

enous injection of 3 � 1011 VP per kilogram ofdMMP8 into 8-week-old CCl cirrhotic animals pro-

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oked a substantial, albeit transient, expression ofMP-8 mRNA transcripts, which could be detected byT-PCR. Hepatic levels of the AdMMP8-directedMP-8 mRNA transcripts were measured at 7 days of

ransduction and varied between individual subjectsn 10), but the same pattern of expression was ob-erved repeatedly in various experiments (Figure 2A).ontrol cirrhotic animals injected with AdGFP did notxpress MMP-8 mRNA transcripts.

igure 2. (A) PCR products of MMP-8 and hypoxanthinephospho-riboreated with AdGFP (lanes 2–4) or AdMMP8 (lanes 5–9) in the presensample from AdMMP8-treated liver in the absence of Moloney murinCR was performed with specific primers for human MMP-8 and HPRTs a size marker. (B) Representative liver sections of cirrhotic rats afdenoviral vector injection. MEC was observed with Masson trichrome

s evident before and after 14 days of AdMMP8 treatment as compaeversion. Twenty microscopic fields per liver biopsy sample from alQwin Leica) to measure fibrosis. Statistical analysis was performedchieved in the MMP-8 group (P � 0.005) compared with nonresponetalloproteinase-8 activity by a specific ELISA in liver homogenateeterminations.

Reversion of CCl4-Induced Cirrhosis by aSingle AdMMP8 Injection via the Iliac Vein

Computer-assisted analyses showed that 8 of 10dMMP8-treated rats had a variable, yet remarkable,egree (30%–60%) of hepatic fibrosis resolution by day4 after adenovirus vector administration (Figure 2B and). It is worth mentioning that each animal was its ownontrol. A liver biopsy sample was taken by laparotomy

nsferase (HPRT) of total RNA extracted from livers of cirrhotic ratsMoloney murine leukemia virus reverse transcriptase. Lane 1 showskemia virus reverse transcriptase in a complementary DNA reaction.A molecular 100-bp DNA ladder (Invitrogen, Carlsbad, CA) was usedweeks of CCl4 administration and AdMMP8 (rats A and B) or AdGFPing (50�). The change in the amount of extracellular matrix material

ith AdGFP-treated animals. (C) Quantitative measurement of fibrosisals were counted and analyzed with computerized Image program

8 responding AdMMP8-treated animals (8 of 10). Significance wasanimals treated with AdGFP and saline. (D) Determination of matrixcirrhotic rats treated as indicated. Values indicate the mean of 3

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1128 SILLER-LOPEZ ET AL. GASTROENTEROLOGY Vol. 126, No. 4

t the moment of adenovirus infusion, and bleeding wastopped with Gel-Foam application. A second compara-ive biopsy sample from the same rat liver was obtainedt the end of treatment, when all animals were killed.uantitative data shown in Figure 2C show that animals

njected with AdGFP (5 of 5) did not show a reductionn either liver fibrosis or cell necrosis. A characteristichronic inflammatory stage was also present. Controlirrhotic animals given saline showed only a minor de-rease in fibrosis, confirming our data on the specificity ofollagenase activity on hepatic ECM degradation. Quan-itative data gathered from animals that responded toreatment showed that results were statistically signifi-ant (P � 0.005). Furthermore, levels of MMP-8 proteinxpression were determined by ELISA and are shown inigure 2D. Liver extracts from AdMMP8-transducedirrhotic animals showed a mean value of approximately50 pg/mL, whereas liver extracts from cirrhotic ratsnjected with the irrelevant adenovirus vector AdGFPontained negligible activity.

Degradation of fibrotic tissue could also be takinglace via activation of latent tissue gelatinases (Figure 3).e measured the expression of MMP-2, MMP-3, and

IMP-1 in liver homogenates from rats made cirrhoticith chronic CCl4 intoxication and BDL for 4 weeks afterdMMP8 iliac vein administration. MMP-2 specifically

igure 3. MMP and TIMP-1estern blot analysis. Liver ho-ogenates from control andxperimental animals were an-lyzed for MMP-2, MMP-3, andree TIMP-1. Representativeamples are shown. (A) MMP-2hows a statistical increaseP � 0.001) in the CCl4/MMP-8roup. (B) MMP-3 defined aarked augmenting tendency

n the treated groups; however,o statistical significance waschieved in any group. (C) FreeIMP-1 increased in groups in-ected with AdMMP8 for bothodels of experimental cir-

hosis. Statistical significanceas present only for the BDL-MP-8 group (P � 0.001).M, CCl4 group administereddMMP8; CG, CCl4 group withdGFP; LM, BDL group treatedith AdMMP8; LG, BDL group

njected with AdGFP; N, nor-al rat.

egrades collagen type IV, and other collagens to a lesseregree.27,28 MMP-2 (Figure 3A) was clearly increased inoth CCl4-induced and BDL-induced experimental cir-hosis animals treated with AdMMP8. Nonetheless, onlyn the CCl4 model was statistical significance achievedP � 0.001). Although there was a small up-regulationn MMP-3 in AdMMP8-treated animals (Figure 3B),here was no statistical significance in any group.IMP-1, which is known to impede MMP’s activity, wasugmented in its free form (28 kilodaltons) in liverxtracts from rats of the experimental cirrhosis group andhe AdMMP8-treated group, but not in the AdGFProup, displaying statistical significance in the BDLodel (P � 0.001; Figure 3C).We also provide mechanistic insights on gene expres-

ion for collagen I, III, and IV; TGF-�; and MMP-9.iver samples from AdMMP8-treated BDL cirrhotic ratsad collagen type I, III, and IV slightly decreased, butith no statistical significance. TGF-� was diminished,

s opposed to MMP-9, which was increased. This isonsistent with protein data obtained in the Westernlot analysis (Figure 4).Decreased fibrosis correlated with improvements in

scites and functional hepatic tests. Severe (�7% of bodyeight) accumulation of peritoneal fluid, an important

linical manifestation of advanced hepatic cirrhosis, was

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etected in all animals treated with AdGFP (5/5). Twoirrhotic animals out of 10 injected with AdMMP8howed moderate ascites (�7% of body weight), whereas

had only trace amounts at the end of 14 days afterdMMP8. An important rearrangement of the hepaticarenchyma and hyperchromatic nuclei was also noted,eflecting a brisk liver cell regeneration that was con-rmed with gene expression for HGF by RT-PCR anal-sis. Figure 5A shows that HGF gene expression wasp-regulated in the cirrhotic rats treated with AdMMP8,ut not in AdGFP-treated cirrhotic animals. These dataorrelate well with the immunohistochemical findingshown in Figure 5B with anti-PCNA antibody staining.iver sections from AdGFP-treated animals did not showignificant liver cell regeneration.

Reduction of hepatic fibrosis resulted in morphologi-al improvement, as can be seen in Figure 5C, where amooth hepatic texture in normal and AdMMP8-treatedat livers is conspicuous, as compared with the rough andranular liver surface from AdGFP-injected animals.urthermore, these findings were accompanied by a clearmprovement in collateral circulation and gastric varicesFigure 5D), suggesting diminished intrahepatic bloodressure in animals injected with AdMMP8.

We wanted to test our proof of concept in a differentirrhosis model that resembles human secondary biliary

brosis. Thus, rats undergoing complete bile duct occlu-ion present progressive and aggressive fibrosis in theirtual absence of inflammation and necrosis. Ten ratsere subjected to ligation and sectioning of the commonile duct, and liver biopsy samples were obtained fromach rat before BDL ligation and at every step afterigation. At the end of BDL (4 weeks), all rats had aramatic increase in liver fibrosis as compared with base-ine fibrosis (Figure 6) and were subjected to a biliodi-estive anastomosis to drain the clogged bile and elim-nate the fibrogenic stimulus. Rats were left at this stageor 7 days. The hepatic fibrosis index, however, remainedncreased to a similar extent in all animals. Five rats werehen injected via the iliac vein with 3 � 1011 VP perilogram of AdMMP8, and 5 rats were injected with 3 �011 VP per kilogram of irrelevant adenoviral vectorAdGFP) as control. The 10 rats were kept under carefulurveillance for 10 more days while they were monitoredor overall health status, and they were then killed. Atatistically significant decrease in fibrosis (45%; P �.05) was noted in rats injected with AdMMP8, but noecrease in the fibrosis index was observed in the 5nimals injected with AdGFP. The biochemical deter-inations of hydroxyproline correlated with the dimin-

shed fibrosis as measured by image morphometric anal-sis (Figure 6B and C).

Figure 4. (A) RepresentativeRT-PCR products of hypoxan-thinephospho-ribosyltrans-ferase and genes involved inECM turnover from livers ofLDB cirrhotic rats treated withAdGFP (LG; lanes 1 and 2) orAdMMP8 (LM; lanes 3 and 4).A 100-base pair (bp) DNA lad-der or �X174 was used as asize marker (MW). (B) Densi-tometric data obtained from 3different experiments areshown as mean � SD. MMP-9and TGF-�1 expression were in-versely expressed afterAdMMP8 treatment. HPRT, hy-poxanthinephospho-ribosyl-transferase; Col, collagen.

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1130 SILLER-LOPEZ ET AL. GASTROENTEROLOGY Vol. 126, No. 4

DiscussionA recent growing body of evidence suggests that

ene therapy with adenovectors as gene carriers may besed efficiently and safely in rats, even in the presence of

igure 5. (A) RT-PCR from liver tissue for hypoxanthinephospho-ribo-yltransferase (HPRT) and HGF. Representative samples from controlnd experimental groups were loaded in agarose gel. Lane 1: molec-lar marker �X174. Lanes 2–4: BDL rats with AdGFP. Lanes 5 and 6:DL rats treated with AdMMP8. (B) Immunohistochemistry on liverlides from control and treated rats with anti-PCNA antibody waserformed to determine hepatocyte proliferation. Hepatocytes withuclear PCNA immunostaining after AdMMP8 treatment were morebundant as compared with livers from AdGFP-administered cirrhoticats. (C) Macroscopic view of normal and AdGFP- and AdMMP8-treatedirrhotic rat livers 14 days after adenovirus administration. A smoothepatic texture in normal and AdMMP8-treated rat livers is clearlyeen, as compared with a rough and granular liver surface fromdGFP-injected animals. (D) A notable improvement in collateral cir-ulation and gastric varices is present (circle).

evere liver disease.11,29,30 The toxicity of adenovirusectors injected into cirrhotic rats is an issue we haveddressed before. We have previously reported that dosesn the order of 1012 VP per kilogram are highly toxic forirrhotic rats, but cirrhotic rats could tolerate doses inhe range of 1011 VP per kilogram.14 Thus, a dose of 3 �011 VP per kilogram was selected to conduct thesexperiments. Obviously, we do not intend to use thishuttle vector to carry genes in human cirrhotic livers inight of all the findings and reports, specifically the Jesseelsinger affair. However, we think we have proven ouroint: that is, we have established our proof of concept inexperimental models resembling human cirrhosis.For clinical scenario application, it must be considered

hat most of the human population has been exposed toild-type adenovirus (especially the Ad2 and Ad5 sero-

ypes used for gene therapy vectors). In patients, theagnitude of the immune response is determined by

reexisting antibody titers and is modified by the routef administration, but it is not dose-response depen-ent.31 A preexisting immune response is expected toiminish adenovector transduction and to produce anncreased immune response that must be considered. It isbvious, then, that a more suitable and safe vector muste designed and developed for use in this particularuman disease.Even so, we have shown that a single dose of a first-

eneration recombinant adenoviral vector carrying a geneor a collagen-degrading enzyme (MMP-8) reversed ex-ensive liver fibrosis, stimulated liver cell proliferation,nd resulted in stabilized functional hepatic tests and theisappearance of abnormal gastric circulation and ascites.n this article, we have confirmed and extended previousbservations, including ours,11,29,30 in the sense thatystemic administration of adenoviral vectors preferen-ially targets livers, even functionally damaged livers,nd that doses in the order of 1011 VP per kilogram areell tolerated by cirrhotic rats.14 Thus, we have shown

hat enough healthy tissue is available for vector trans-uction that sufficed to induce a repairing response. Inhis study, we observed that regardless of the onset of ahronic liver disease in cirrhotic animals at the momentf “therapeutic” vector administration, MMP-8 trans-ene expression was effective and significant. These re-ults, along with increasing compelling evidence,11,29,30

mply that transient or regulated expression of a geneherapy targeted to the hepatic gland may be useful as aherapeutic strategy even in the presence of severe, lin-ering hepatic disease.The discovery of processes that regulate the extent of

brogenesis and the mechanisms governing ECM degra-

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April 2004 CIRRHOSIS IMPROVEMENT BY MMP–8 GENE TRANSFER 1131

ation have been slow to emerge. Impairment of anrgan’s ability to repair itself after a trauma or a sus-ained injury will determine the onset of a fibrogenicesponse, invariably leading to abated functioning.32

Several mechanisms control ECM synthesis and ECMegradation. Degradation of ECM proteins is mainlyediated by MMPs, which represent a proteinase family

hat requires zinc ions for activity. MMPs are synthesizedn demand and secreted, in most cases, as nonactiveroenzymes that are in turn activated by various pro-eases, such as plasmin11,33,34 and stromelysin.35 In thisrticle, we found by Western blot analysis an increase inhe production of MMP-2, MMP-3, and free TIMP-1 iniver extracts from cirrhotic rats treated with AdMMP8.

igure 6. Induction of fibrosis reversion by 3 � 1011 viral AdMMP8 parections of cirrhotic rats after bile duct ligation, bile drainage by biliodasson trichrome staining (100�) shows cirrhotic nodules surroundowever, animals treated with AdMMP8 showed only thin fibrous ba

issue deposition at every step and after AdGFP or AdMMP8 adenovirandom fields by an automated image analyzer. Values are presentediver tissue at the end of treatment (P � 0.05). Ad, adenovirus; BD,

ree TIMP-1 was determined as an indirect measurementf active uncomplexed MMPs. Mechanisms other thanroteolysis can render MMPs activation; it has beenhown that stoichiometric activation of human skin fi-roblast procollagenase can be performed by factorsresent in human skin and rat uterus.36 In this study, wechieved high AdMMP8-driven MMP-8 collagenase pro-uction. Such an enzyme had a biochemical performances the naturally occurring protein and induced a decreasen liver fibrosis in 2 different experimental cirrhosis

odels. The higher concentration of MMP-8 inside theell as compared with the extracellular culture mediaight indicate that MMP-8 could be stored in specific

ranules within the transduced cells, similarly to the

per kilogram in rat cirrhosis after prolonged bile obstruction. (A) Liverive anastomosis, and AdMMP8 or AdGFP adenoviral vector injection.thick fibrotic bands (blue) even at the end of treatment with AdGFP.

consisting of connective tissue proteins. (B) Percentages of fibrousctor infusion. Determinations of fibrous tissue were performed in 20ean � SD. (C) Biochemical determination of hydroxyproline (HyP) inuct.

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1132 SILLER-LOPEZ ET AL. GASTROENTEROLOGY Vol. 126, No. 4

aturally occurring storage of MMP-8 in neutrophileukocytes. Furthermore, consistent with previous find-ngs with COS cells transfected with an MMP-8 plasmid,here was also a significant and constitutive secretion of

MP-8 into the culture supernatant.13 The level oflycosylation of MMP-8 has been associated with thentracellular storage or release of the enzyme to thextracellular compartment. In neutrophils, MMP-8 isighly glycosylated and remains stored in intracellularranules, whereas poorly glycosylated recombinantMP-8 from plasmid-transfected COS cells is released to

he culture supernatant.13,37 The specific intracellularocalization of MMP-8 in HeLa-transduced cells and theegree of glycosylation of the recombinant protein that isroduced remains to be elucidated.Furthermore, our data suggest that MMP-8 is efficiently

onverted to a functional collagenase in vivo, regardless ofhe unelucidated mechanism. This is evident from theollagenolytic activity assay, in which type I collagen isegraded in the absence of APMA (Figure 1C).

Administration of the MMP-8 cognate gene in cirrhoticnimals induced amelioration of hepatic cirrhosis. Remod-ling of ECM excessively deposited in cirrhotic livers iserformed by a tilted positive balance of artificially drivenMP-8 getting activated inside the fibrotic liver. This is

nhanced by naturally occurring MMP-2 and MMP-3,hich seem to be up-regulated by MMP-8 treatment.onetheless, other fibrinolytic mechanisms might also be

aking place. Alteration of MMP expression plays a majorole in liver fibrogenesis and cirrhosis. Furthermore, anotherevel of control of MMP activity is achieved through itshysiological inhibitors (TIMPs) by stabilizing the proen-yme and inhibition of the active species.38,39 TIMP-1 isble to block every MMP. The TIMP-1 increase we foundn its free form suggests that a pool of metalloproteasesother than MMP-8) were rendered active and were ready toegrade the fibrotic scar.

The fact that we found increased liver cell proliferationi.e., abundant hyperchromatic nuclei, and then mea-ured by immunohistochemistry with the proliferationarker [PCNA] dispersed throughout the hepatic paren-

hyma and gene expression of HGF in MMP-8 treatedats) suggests that a change—not only in the amount,ut also in the nature—of the ECM is ongoing. Collagendecreases have been shown to enhance liver restorationy inducing HSC apoptosis and hepatocyte regeneration.ype I collagen perpetuates or promotes the activatedhenotype of HSC.40 Besides, direct contact of epithelialells with collagen type I takes them out of the cell cycle;owever, once they contact partially degraded collagen Iediated by � , integrin reenters the cell cycle. Be-

3

ause collagen type I is the main MMP-8 substrate, itsegradation represents a favorable event that contributeso restitution of hepatic cell population, as corroboratedy our findings. Also, it has been shown in liver-regen-rated rats after partial hepatectomy that the synthesis ofCM might play an important role in reestablishing theuiescent and differentiated phenotype of hepatocytes.41

everal in vitro studies showed that ECM39 modulateshe phenotype of hepatocytes. Therefore, the notion thatn AdMMP8-treated animals a different kind of ECM isresent and the reasoning that potent growth factors areeleased in situ (such as HGF), resulting in cell prolif-ration, seem plausible. In this line of evidence, HGFas mainly embedded in the ECM of liver, spleen, andidney bound to matrix proteins including heparan sul-ate, thrombospondin, and collagen types I, III, IV, V,nd VI.42

The effect of ECM remodeling phenomena on theverall clinical status of AdMMP8-treated cirrhotic ani-als deserves further consideration. It is clear that liver

nternal mechanisms caused improved hepatic syntheticunctioning, as reflected in the disappearance of ascitesnd the reestablishment of transaminases (alanine ami-otransferase and aspartate aminotransferase).In addition, more compelling evidence of liver func-

ion improvement was a notable decrease of collateralastric varices, which is equal to restored intrahepaticlood pressure in animals treated with the therapeuticdMMP8 vector. Effective therapies for alcohol-induced

iver cirrhosis and cirrhosis resulting from bile obstruc-ive disorders are lacking, and the disease is increasing;herefore, a gene therapy approach is attractive. Weelieve that therapy with the MMP-8 gene holds promiseor use in a clinical setting.

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Received December 19, 2001. Accepted December 18, 2003.Address requests for reprints to: Juan Armendariz-Borunda, M.D.,

nstitute for Molecular Biology in Medicine and Gene Therapy, CUCS,niversity of Guadalajara, Apdo. Postal 2-123, Guadalajara, Jal 44281,

exico. e-mail: armendbo@cucs.udg.mx; fax: (52) 33-3618-7473.