GASTROENTEROLOGY 2007;133:91107

Signicance and Therapeutic Potential of Endothelial Progenitor Cell Transplantation in a Cirrhotic Liver Rat Model
TORU NAKAMURA,*, TAKUJI TORIMURA,*, MASAHARU SAKAMOTO,*, OSAMU HASHIMOTO,*, EITARO TANIGUCHI,*, KINYA INOUE,*, RYUICHIRO SAKATA,*, RYUKICHI KUMASHIRO,* TOYOAKI MUROHARA, TAKATO UENO, and MICHIO SATA*
*Division of Gastroenterology, Department of Medicine, Kurume University School of Medicine, Kurume, Fukuoka; Liver Cancer Division, Research Center for Innovative Cancer Therapy and Center of 21st Century COE Program for Medical Science, Kurume University, Kurume, Fukuoka; and Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan CLINICALLIVER, PANCREAS, AND BILIARY TRACT

Background & Aims: We investigated whether endothelial progenitor cell (EPC) transplantation could reduce established liver brosis and promote hepatic regeneration by isolating rat EPCs from bone marrow cells. Methods: Recipient rats were injected intraperitoneally with carbon tetrachloride (CCl4) twice weekly for 6 weeks before initial administration of EPCs. CCl4 was then readministered twice weekly for 4 more weeks, and EPC transplantation was carried out for these same 4 weeks. Results: At 7 days in culture, the cells expressed Thy-1, CD31, CD133, Flt-1, Flk-1, and Tie-2, suggesting an immature endothelial lineage. Immunohistochemical analyses showed uorescent-labeled, transplantation EPCs were incorporated into the portal tracts and brous septa. Single and multiple EPC transplantation rats had reduced liver brosis, with decreased 2-(I)procollagen, bronectin, transforming growth factor- , and -smooth muscle actin-positive cells. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area, in accordance with EPC location. Real-time polymerase chain reaction analysis of multiple EPC-transplantation livers showed signicantly increased messenger RNA levels of matrix metalloproteinase (MMP)-2, -9 and -13, whereas tissue inhibitor of metalloproteinase-1 expression was signicantly reduced. Expression of hepatocyte growth factor, transforming growth factor- , epidermal growth factor, and vascular endothelial growth factor was increased in multiple EPC-transplantation livers, while hepatocyte proliferation increased. Transaminase, total bilirubin, total protein, and albumin levels were maintained in EPC-transplantation rats, signicantly improving survival rates. Conclusions: We conclude that single or repeated EPC transplantation halts established liver brosis in rats by suppressing activated hepatic stellate cells, increasing matrix metalloproteinase activity, and regulating hepatocyte proliferation.

hepatitis virus B and hepatitis virus C infection, alcohol abuse, or nonalcoholic steatohepatitis.13 Advanced liver brosis results in cirrhosis, liver failure, and portal hypertension, and this condition often requires liver transplantation. Liver transplantation may be the only alternative to treat patients with a heavily damaged liver, as in cases of severe cirrhosis. Liver transplantation improves both survival and quality of life. However, there are only a limited number of donor livers available for patients.1,4 Therefore, it is very important to investigate appropriate new therapies. Several recent promising studies have shown that hematopoietic stem cells from bone marrow (BM) have the capacity to differentiate into a variety of nonhematopoietic cell tissues such as liver, heart, and brain.511 Even multiorgan differentiation was demonstrated from hematopoietic stem cells and from the recently described multipotent adult progenitor cells.12,13 Perfusion of these cells is one potential approach to promoting brosis resolution and hepatic regeneration. Endothelial progenitor cells (EPCs) are mobilized from BM and incorporated into sites of vascular disorders, at which they aid in neovascularization.14 17 Neovascularization involves angiogenesis, ie, formation of new blood vessels via sprouting of preexisting mature endothelial cells, and also vasculogenesis, which is formation of blood vessels by differentiation of EPCs.14,18,19 We recently reported that EPC transplantation signicantly enhanced vascularization and improved survival rates after acute liver injury in mice.20 However, it remains unclear whether EPCs can inhibit brogenesis in vivo and
Abbreviations used in this paper: -SMA, -smooth muscle actin; BM, bone marrow; CCl4, carbon tetrachloride; ECM, extracellular matrix; EGF, epithelial growth factor; EPC, endothelium progenitor cell; Flk-1, fetal liver kinase-1; Flt-1, fms-like tyrosine kinase-1; HGF, hepatocyte growth factor; HSC, hepatic stellate cell; MMP, matrix metalloproteinase; RAECs, rat aortic endothelial cells; TAA, thioacetamide; TGF, transforming growth factor; Tie-2, tyrosine kinase with Ig and EGF homology domains-2; TIMP, tissue inhibitor of metalloproteinase; VEGF, vascular endothelial growth factor. 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.03.110

t is widely recognized that the development of liver brosis is intimately associated with the progression of chronic liver disease caused by agents such as chronic

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whether EPC transplantation could be effective against established liver brosis. We therefore investigated the antibrogenic and regenerative effects of EPC transplantation in carbon tetrachloride (CCl4)- and thioacetamide (TAA)-induced cirrhosis.

cent marker PKH26-red (Sigma Chemical Co., St. Louis, MO) following the manufacturers instructions before EPC transplantation into rats, as described previously.20

Materials and Methods Animals

The study was conducted using 6-week-old male Wistar rats having an approximate body weight (BW) of 120 150 g. Wistar rats were purchased from CLEA Japan Inc. (Shizuoka, Japan). Animals were maintained in temperature-controlled rooms (21C 2C) under a 12/12hour dark/light cycle and allowed food (standard laboratory chow) and water ad libitum.

Experimental Conditions and Transplantation of EPCs

The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Kurume University School of Medicine. Cirrhosis was induced by 2 different methods. For the CCl4 (Wako, Osaka, Japan)-treated model, phenobarbital sodium (Wako; 35 mg/dL) was added to the drinking water for 1 week. The rats then received intraperitoneal injections of 50% CCl4 (10 mg/kg body weight) twice weekly for 6 weeks, as described previously.24 After 6 weeks of CCl4 injections, the rats were divided randomly into 3 treatment groups: (1) saline infused (n 26); (2) EPC treated (n 20) once weekly; and (3) EPC treated (n 18) on day 43 only. EPC (recipient) rats received EPCs via the tail vein (3.0 106 cells/rat). Following EPC transplantation, CCl4 administration was continued twice weekly for another 4 weeks. Rats also received EPCs or a saline injection once weekly over this same 4-week period. The rats were killed after this 10-week period of CCl4 injection. For the TAA (Wako)-treated model, rats were injected intraperitoneally with TAA (200 mg/kg body weight) twice weekly for 6 weeks, as described previously.25 After 6 weeks of TAA injections, the rats were divided randomly into 2 treatment groups: (1) saline infused (n 15) and (2) EPC treated once weekly (n 15). EPC (recipient) rats received EPCs via the tail vein (3.0 106 cells/rat). Following EPC transplantation, TAA administration continued twice weekly for another 3 weeks. Rats also received EPCs or a saline injection once weekly over this same 3-week period. The rats were killed after this 9-week period of TAA injection. To determine whether the living EPCs were effective, we performed irradiated EPC transplantation. EPCs were irradiated with 100 mJ/cm2 ultraviolet (UV) radiation for 3 hours in a Spectrolinker XL-1000 UV crosslinker (Spectronics Corporation, Westbury, NY), and the cells were collected and underwent transplantation using the same experimental methods as for EPCs. To clarify whether transplanted (PKH26 ) cells were tracked in cirrhotic liver, we performed sex-mismatched EPC transplantations from male donor rats into female recipients; female rats treated for 6 weeks with CCl4 received male EPCs (3.0 106 cells/rat). The rats were killed 1 week after this EPC transplantation, and liver sections were analyzed using uorescent in situ hybridization (FISH) to detect the Y chromosome.

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Isolation of Mononuclear Cells

EPC-like mononuclear cells (MNCs) in the BM were quantied. Donor rats were killed to collect BM cells from the right and left femurs by ushing the BM cavity with heparinized saline.21 The resulting cell suspension was centrifuged over a Ficoll step gradient (density, 1.077 g/mL) (Ficoll-Histopaque 1077; Sigma-Aldrich, Steinheim, Germany) at 2000 rpm for 30 minutes. The interface fraction was collected, and mononuclear cells were then resuspended in cell culture medium as described below. They were then plated on 2% gelatin-coated 100-mm plastic dishes.

Culture of EPCs
Medium 199 containing 20% fetal bovine serum (FBS; Biowest, Caille, France), bovine pituitary extracts (Biomedical Tech. Inc., Stoughton, MA) to stimulate cell growth, heparin (100 g/mL), and antibiotics (GIBCO, Auckland, NZ) were used for cell culture. Attached (AT) cells were allowed to develop into EPC for 7 days of culture, at which time culture dishes were washed with phosphate-buffered saline (PBS), and ow cytometry was performed as described previously.14,20,22 EPCs attached to dishes were treated with trypsin-EDTA solution (SigmaAldrich) for 5 minutes, and detached cells were collected. After washing with PBS, EPCs were suspended in PBS at a density of 3.0 106/1 mL and injected into rats as described below.

Primary Culture of Rat Aortic Endothelial Cells

Rat aortic endothelial cells (RAECs) were isolated from rat aorta and then subcultured as reported previously23 and were then grown in Dulbeccos modied Eagle medium (DMEM) (Sigma-Aldrich, Ayershire, United Kingdom) containing 10% fetal bovine serum (FBS) and P-S solution (GIBCO).

Cell Labeling
Experiments were carried out to detect EPCs as follows: the cultured cells were labeled with red uores-

Flow Cytometry
EPCs on day 7 of culture (n 5) were analyzed by uorescence-activated cell sorting (FACS; FACS SCAN

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owcytometer, Becton-Dickinson, San Diego, CA). EPCs were isolated from culture plates after incubation with PBS containing 1 mmol/L EDTA (pH 7.4) for 15 minutes at 37C. After xation, the cells were incubated with primary antibodies to CD45 (Becton-Dickinson), Thy-1 (CD90; Becton-Dickinson), CD31 (Becton-Dickinson), and fetal liver kinase-1 (Flk-1) (Abcam, Cambridge, United Kingdom), as described previously.14,22 The cells were washed with PBS, incubated with rabbit anti-mouse FITC-conjugated antibody, and then analyzed using Quad Statistics of CellQuest software (Becton-Dickinson).

Fluorescence In Situ Hybridization for Y Chromosome

Fluorescence in situ hybridization (FISH) to detect the Y chromosome was performed according to the STARFISH manufacturers protocol using STARFISH Rat 12/Y Paints (Y: FITC, 12: Biotin) (Cambio, Cambridge, United Kingdom). The Cambio paints bind chromosomes Y and 12, respectively, allowing their differentiation, because they contain homologous regions. After hybridizing with chromosome 12 and Y probes, sections were incubated with anti FITC-Alexa 594 (for chromosome Y detection; Invitrogen, Carlsbad, CA) and anti-streptavidin-Cy3 (for chromosome 12 detection; Amersham Pharmacia, Buckinghamshire, England) as secondary antibodies. The FISH images were obtained using a confocal microscope with settings adjusted to split the Alexa 594 and Cy3 signals. Nuclei were labeled using DAPI staining.

the percentage of regenerative hepatocytes with Ki-67positive nuclei relative to the total number of hepatocytes in randomly selected sections (under 100 magnication, 6 elds for each of 5 rats, as above), as described previously.26,27 For the immunouorescence examination, liver tissues were embedded in OTC compound and snap frozen in liquid nitrogen. Five-micrometer cryosections were xed with 4% paraformaldehyde or acetone and incubated with 5% bovine serum albumin (BSA) in PBS and then incubated with primary antibody overnight at 4C. Slides were washed thoroughly, incubated with FITC-conjugated secondary antibodies (1: 100; DAKO) for 30 minutes at room temperature and then washed and mounted with TO-PRO-3 iodide (1: 4000, Invitrogen, Carlsbad, CA) to label the nuclei. Primary antibodies were as follows: anti-CD31 (1:100; BD Biosciences), anti-cytokeratin 19 (1:50; DAKO), anti- -SMA (1:100), anti-matrix metalloproteinase (MMP)-2 (1:100; Santa Cruz Biotechnology), antiMMP-9 (1:250; Daiichi Fine Chemical, Toyama, Japan), and anti-MMP-13 (1:100; Santa Cruz Biotechnology). Four-color imaging was performed in 10 border zone regions for each sample (z-series, 63 oil magnication, Zeiss LSM-510 Meta Confocal Microscope; Carl Zeiss Inc, Jena, Germany) and analyzed with Zeiss LSM Image Browser software version 3.5 (Carl Zeiss Inc).

Gelatin Zymography
Frozen liver tissues were homogenized in buffer (50 mmol/L Tris-HCl; pH 7.6, 150 mmol/L NaCl, 0.1% SDS, 1% Deoxycholate Na, 1% NP-40, 1% Triton X-100), and the protein concentration was determined using the DC Bio-Rad assay (Bio-Rad Laboratories, Hercules, CA). Triplicate samples at each time point were pooled (40 g total) and resolved on a 10% SDS-PAGE gel containing 0.1% gelatin. Gels were placed in 2.5% Triton X-100 for 30 minutes and then incubated for 18 hours in buffer containing 50 mmol/L Tris, pH 7.5, 5 mmol/L CaCl2, then 40 mmol/L NaN3, and stained with coomassie blue R-250. Gels were scanned using an EPSON Ofrio ES-10000G scanner (EPSON, Tokyo, Japan) and analyzed using Adobe Photoshop 5.5 (Adobe System Inc., San Jose, CA) in gray scale at 600 dpi. Images were analyzed using a NIH Image analyzer.

Immunohistochemical Analysis
Liver tissues were xed in buffered formalin, embedded in parafn, cross sectioned, and mounted on silanized slides (Dakocytomation, Kyoto, Japan). Tissue sections were either subjected to Azan-Mallory staining or were analyzed histochemically using antibodies against collagen type I (1:1000; LSL), bronectin (1:200; Chemicon, Temecula, CA), -smooth muscle actin (SMA) (1:100; DAKO, Kyoto, Japan), transforming growth factor (TGF)- (1:100; Santa Cruz Biotechnology), and Ki-67 (1:50; DAKO, Kyoto, Japan). Immunoreactivity was visualized using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DOJINDO; Wako), as described previously.26 For semiquantitative analysis of brosis, the blue-stained area in the AzanMallory stained sections was measured on a video screen display (Nikon Digital sight DS-L1, Tokyo, Japan) using a NIH image analyzer (under 200 magnication, 6 elds for each of 5 rats, ie, a total of 30 elds for each group) by a technician who was blinded to the treatment regimens. The Ki-67 labeling index (Ki-LI) represented

Film In Situ Zymography

The fresh specimens of CCl4-treated liver tissues were embedded without xation in OTC compound and snap frozen in liquid nitrogen. Four-micrometer cryosections were mounted on gelatin lms coated with 7% gelatin solution (Wako). The lms with sections were incubated for 10 hours at 37C in a moisture chamber and stained with Biebrich Scarlet (Wako). The gelatin in contact with the proteolytic areas of the sections was digested, and zones of enzymic activity were indicated by

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negative staining. In addition, we used gelatin-coated lm containing 1, 10-phenanthroline (MMP-PT in situ Zymo-Film; Wako) to distinguish MMP activity from other proteinase activity in the tissue. The digested areas in the sections were compared with serial sections stained with H&E.

Table 1. SYBR Green Real-Time Quantitative PCR Primer Sequences

Target gene Primer Sequence

Total RNA Extraction, Complementary DNA Synthesis, and Reverse-Transcription Polymerase Chain Reaction
For total RNA isolation, cultured cells or 100 mg of liver tissue were extracted according to the Isogen method (Nippon Gene, Tokyo, Japan). RNA was quantied by spectrophotometry. Complementary DNA (cDNA) was synthesized using 2 g total RNA. The 20 L reverse-transcription (RT) reaction consisted of 5X rst strand buffer, 0.5 mmol/L dNTP, 50 nmol/L random primers, and 20 U SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). RNA and primers were mixed and denatured by heating at 70C for 10 minutes, and the RT reaction mixture then was incubated for 30 minutes at 50C, followed by 15 minutes at 70C. A notemplate control was processed for each experiment, establishing the absence of genomic contamination of the samples. The resulting cDNA then was amplied by polymerase chain reaction (PCR) with primer pairs specic for rat CD133, fms-like tyrosine kinase-1 (Flt-1), Flk-1, tyrosine kinase with Ig and EGF homology domains-2 (Tie-2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Flt-1, Flk-1, and Tie-2, 30 cycles; CD133, 35 cycles; and GAPDH, 28 cycles). PCR products were resolved in 1.5% to 1.8% agarose gels and visualized by ethidium bromide staining and ultraviolet transillumination. PCR cycle conditions for CD133 were 94C for 30 seconds, 60C for 30 seconds, and 72C for 1 minute; for Flt-1, 94C for 1 minute, 59C for 1 minute, and 72C for 1 minute; for Flk-1, 94C for 1 minute, 57C for 1 minute, and 72C for 1 minute; for Tie-2, 94C for 1 minute, 59C for 1 minute, and 72C for 1 minute; and, for GAPDH, 94C for 30 seconds, 60C for 30 seconds, and 68C for 2 minutes. Primer pair sequences were as follows: CD133, 5=-GCC ACC GCT CTA GAT ACT GC-3= and 5=-TGT TGT GAT GGG CTT GTC AT-3= (520 base pair [bp]); Flt-1, 5=-CAG AAG AGG ATG AGG GTG TCC A-3= and 5=-CCT AAT GCC AAA TGC CGA AGC C-3= (386 bp); Flk-1, 5=-GGT GAT CCC ATG CCG AGG-3= and 5=-TTT GAG GAC GGG AAT TGC CAG-3= (432 bp); Tie-2, 5=-GGG CAA AAA TGA AGA CCA GCA C-3= and 5=-GCA TCC ATC CGT AAC CCA TCC T-3= (525 bp); and GAPDH, 5=-ACC ACA GTC CAT GCC ATC AC-3= and 5=-TCC ACC ACC CTG TTG CTG TA-3= (452 bp).

2-(I)Forward 5=-ATG TTC AGC TTT GTG GAC CT-3= procollagen Reverse 5=-CAG CTG ACT TCA GGG ATG T-3= Fibronectin Forward 5=-AGA CTG CAG TGA CCA CCA TCC-3= Reverse 5=-CAA TGT GTC CTT GAG AGC ATA GAC-3= TGFForward 5=-TTG CCC TCT ACA ACC AAC ACA A-3= Reverse 5=-GGC TTG CGA CCC ACG TAG TA-3= -SMA Forward 5=-CGA AGC GCA GAG CAA GAG A-3= Reverse 5=-CAT GTC GTC CCA GTT GGT GAT-3= EGF Forward 5=-ACT ATA ACG GTG GCT CCA TCC A-3= Reverse 5=-CAG TGT GTT TGT CGG CTA TCC A-3= TGFForward 5=-CAT CAC TGC CCT GGT GGT AGT-3= Reverse 5=-GGA CCT GAC AGC AGT GGA TCA-3= HGF Forward 5=-CCC ACA AGG GCT TTG ATG A-3= Reverse 5=-GCG CAT GTT TTA ATT GCA CAG T-3= VEGF Forward 5=-GGG CTG CTG CAA TGA TGA A-3= Reverse 5=-TGC TGC AGG AAG CTC ATC TCT-3= MMP-2 Forward 5=-CCG AGG ACT ATG ACC GGG ATA A-3= Reverse 5=-CTT GTT GCC CAG GAA AGT GAA G-3= MMP-9 Forward 5=-TGG AAC TCA CAC AAC GTC TTT CA-3= Reverse 5=-TCA CCC GGT TGT GGA AAC TC-3= MMP-13 Forward 5=-GGT TGA GCC TGA ACT GTT TTT GA-3= Reverse 5=-CTC GTA TGC AGC ATC CAC ATG-3= TIMP-1 Forward 5=-AGC CTG TAG CTG TGC CCC AA-3= Reverse 5=-AAC TCC TCG CTG CGG TTC TG-3= TIMP-2 Forward 5=-GCC CTA TGA TCC CAT GCT ACA-3= Reverse 5=-TCT GTG ACC CAG TCC ATC CA-3= 18S Forward Proprietary (Applied Biosystems) Reverse Proprietary (Applied Biosystems)

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SYBR Green Real-Time Quantitative PCR

For the quantitative SYBR Green real-time PCR, 0.6 L of each RT product was used per reaction, and the SYBR Green reaction was conducted using a SYBR Green PCR Core Reagents kit (PE Biosystems, Warrington,

United Kingdom) according to the protocol provided by the manufacturer. Optimization was performed for each gene-specic primer prior to the experiment to conrm that 50-nmol/L primer concentrations did not produce a nonspecic primer-dimer amplication of the signal in the no-template control tube. The primer sequences were designed using Primer Express Software (PE Applied Biosystems, Foster City, CA) and are presented in Table 1. The primers for 18S rRNA were purchased from a commercial vendor. Quantitative PCR was performed on the ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems) by using 3-stage program parameters provided by the manufacturer as follows: 2 minutes at 50C, 10 minutes at 95C, and then 40 cycles of 15 seconds at 95C and 1 minute at 60C. The specicity of the produced amplication products was conrmed by examination of the dissociation reaction plots. A distinct single peak indicated that a single DNA sequence was amplied during PCR. End-reaction products were visualized on ethidium bromide-stained 1.5% agarose gels. Appearance of a single band having the correct molecular size conrmed specicity of the PCR. Results represent 3 independent experiments. All products were sequenced to conrm their identity.27

Western Blot Analysis

Cells were lysed in a lysis buffer (0.1% NP-40, 50 mmol/L HEPES, pH 7.5, 250 mmol/L NaCl, 20

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Figure 1. Characterization of cultured mononuclear cells derived from bone marrow. (A and B) Attached (AT) cells were observed to form clusters similar to blood islands and cord-like structures similar to blood vessels on day 7. (C) Flt-1, Flk-1, and Tie-2 mRNA were expressed both in endothelial progenitor cells (EPCs) and in rat aortic vascular endothelial cells (RAECs), whereas CD133 was detected only in EPCs after 7 days of culture. (DH) Flow cytometric analysis of not AT and AT cells on day 7. Not AT cells were positive for CD45 (46.4% 0.9%; D). AT cells were positive for Thy-1 (67.1% 3.7%; F), CD31 (65.6% 3.2%; G), and Flk-1 (56.9% 2.6%; H) but not for CD45 (2.3% 2.1%; E). Data show the percentage of positive cells (n 5). Original magnication in A, 200; B, 100.

mmol/L EDTA) containing 1 mmol/L phenylmethylsulfonyl uoride and a protein inhibitor cocktail (Sigma, Tokyo, Japan). Cell lysates were incubated at 4C for 10 minutes and claried by centrifugation (12,000 rpm) for 20 minutes at 4C. Frozen liver tissues were homogenized in the same lysis buffer and were centrifuged for 30 minutes at 4C. Supernatants were subjected to protein determination with the DC protein assay kit (Bio-Rad Laboratories), according to the manufacturers instructions. Total cells and tissues lysates were added to an equal volume of 2X sample loading buffer containing 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol and boiled for 10 minutes. Total cell protein (50 g) was loaded onto an SDS-polyacrylamide gel, electrophoresed, and electrically transferred to a Fluorotrans Membrane (Pall Life Science, Ann Arbor, MI). Following transfer, the membrane was blocked for 1 hour in 5% nonfat dried milk

and then incubated overnight at 4C with a primary antibody (MMP-13, 1:2000; and -actin, 1:500; Sigma Chemical Co, St. Louis, MO). Visualization of the protein signal was achieved with a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence procedures according to the manufacturers recommendation (Amersham Pharmacia Biotech, Piscataway, NJ). The protein signals were scanned using an EPSON Ofrio ES-10000G scanner and analyzed using Adobe Photoshop 5.5 in gray scale at 600 dpi. Images were analyzed using a NIH Image analyzer.

Serum Analysis
Biochemical parameters and serum type IV collagen levels were measured using standard clinical methods (Department of Clinical Laboratory, Kurume University Hospital).

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Figure 2. Distribution of transplantation EPCs in cirrhotic rat liver. (A) FISH with rat Y chromosome and rat 12 chromosome probes allowed detection of the Y chromosome. Triple staining for rat Y chromosome (red), rat 12 chromosome (yellow), and nuclei (blue) of liver cryosections from female rats treated with CCl4 for 7 weeks. Female livers were extracted 1 week after male EPC transplantation; nuclei were stained with DAPI. Arrows indicate the transplantation male cells. (B) Y chromosomes (arrows) could be detected by FISH in male cirrhotic rat liver sections used as a positive control. (CF) PKH26 EPCs (red) were found on the vasolateral surface of CD31 (green) blood vessels (12 hours following transplantation; C), within vessel walls (1 day following transplantation; D), near the vessels within the brotic areas (3 days following transplantation; E), and around the portal tracts and brous septa (1 week following transplantation; F) of CCl4-treated livers. (GI) In livers obtained 4 weeks after EPC transplantation, most PKH26 EPCs (red; H and I) were also CD31 positive (green; G and I). (J) PKH26 EPCs (red) were positive for CD31 (green) and formed vessels, whereas they were negative for -SMA (green) and cytokeratin-19 (green) at 4 weeks following transplantation. Arrows indicate PKH26 EPCs. Tissues were stained with TO-PRO-3 iodide to visualize the nuclei (blue). Scale bar, 20 m in CF and J and 100 m in GI.

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Figure 3. Microphotographs of liver sections from CCl4-treated rats (A and C) and TAA-treated rats (B and D). (A and B) Fibrosis was less notable in single and multiple EPC-transplantation than in irradiated EPC-transplantation or saline-infused livers according to Azan-Mallory staining. (C and D) The percentage of the brotic areas was signicantly less in single and multiple EPC-treated livers than in irradiated EPC-transplantation or saline-infused livers (*P .01; n 10). Intact, normal livers; CCl4 6W, livers after 6 weeks of CCl4 treatment; CCl4 10W Saline, saline-infused livers after 10 weeks of CCl4 treatment; CCl4 10W Irradiated EPC, livers undergoing transplantation 4 times with irradiated EPC after 10 weeks of CCl4 treatment; CCl4 10W single EPC, livers undergoing transplantation once with EPC after 10 weeks of CCl4 treatment; CCl4 10W Multiple EPC, livers undergoing transplantation 4 times with EPC after 10 weeks of CCl4 treatment. TAA 6W, livers after 6 weeks of TAA treatment; TAA 9W Saline, saline-infused livers after 9 weeks of TAA treatment; TAA 9W Multiple EPC, livers undergoing transplantation 3 times with EPC after 9 weeks of TAA treatment. Original magnication, 40.

Statistical Analysis
Differences between groups were examined for statistical signicance using the MannWhitney U test and the KruskalWallis nonparametric ANOVA. All data

are expressed as the mean SEM, and P values less than 5% were considered to indicate signicance. Overall survival was estimated according to the KaplanMeier method and compared using the log-rank test.

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Figure 4. (A) Histologic assessment of CCl4-induced liver brosis. Localization of type I collagen (Col. I) and bronectin (FN) was less apparent in multiple EPC-transplantation livers than in irradiated EPC-transplantation or saline-infused livers. In addition, -SMA and TGF- expression was also diminished in multiple EPC-transplantation livers. No signicant difference was seen between irradiated EPC-transplantation and saline-infused livers. CCl4 6W, livers after 6 weeks of CCl4 treatment; CCl4 10W Saline, saline-infused livers after 10 weeks of CCl4 treatment; CCl4 10W Irradiated EPC, livers undergoing transplantation 4 times with irradiated EPC after 10 weeks of CCl4 treatment; CCl4 10W Multiple EPC, livers undergoing transplantation 4 times with EPC after 10 weeks of CCl4 treatment. Original magnications: Col. I and FN, 100; -SMA and TGF- , 200. (B) Quantitative real-time PCR analysis of mRNA expression levels of 2-(I)-procollagen, bronectin, -SMA, and TGFafter treatment for EPCtransplantation (light gray shaded columns) and saline-infused (dark gray shaded columns) liver tissues. Expression of 2-(I)-procollagen, bronectin, -SMA, and TGF- mRNA after CCl4 treatment for 8 10 weeks was signicantly reduced in EPCtransplantation livers compared with saline-infused livers. Results are expressed in arbitrary units (*P .05 compared with saline-infused livers at each time point; n 10). Solid columns showed livers after 6 weeks of CCl4 treatment. Intact, normal livers (open columns).

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Results Identication of BM-Derived MNCs

After 7 days in culture ex vivo, expanded MNCs derived from the BM of healthy rats had attached to culture dishes, in which they formed multiple cell clusters similar to blood islands (Figure 1A) and cord-like structures resembling blood vessels (Figure 1B). These cells displayed properties of endothelial lineage cells. RT-

PCR indicated that these cells expressed mRNA encoding CD133 (a marker for stem/progenitor cells) and other endothelial cell markers such as Flt-1, Flk-1, and Tie-2 (Figure 1C). In mature RAECs, mRNA for Flt-1, Flk-1, and Tie-2 were detected, whereas CD133 mRNA was not. We also performed ow cytometric analyses of endothelial cell-like phenotype cells at day 7 of culture to

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Table 2. Serum Hepatic Enzyme and Type IV Collagen in CCl4-Induced Liver

Type IV collagen (ng/mL) Intact CCl4 6W CCl4 10W CCl4 10W 24.6 50.7 17.1 7.0 13.1 6.4a AST (U/mL) 82 268.5 299.1 191.5 4.1 54.6 54.0 21.6 ALT (U/mL) 45 100 109.8 75.9 6.0 7.8 10.0 6.3a Total bilirubin (mg/dL) 0.07 0.66 0.83 0.2 0.05 0.26 0.22 0.03a Total protein (g/dL) 4.9 4.82 3.82 4.76 0.14 0.33 0.29 0.21a Albumin (g/dL) 2.4 2.0 1.37 1.75 0.05 0.2 0.10 0.09a

saline multiple EPC

W, weeks. aP .05 vs CCl4 10W

saline.

examine the expression of CD45 (a marker for leukocyte common antigen), Thy-1 (CD90, a marker for rat hematopoietic stem cells), CD31, and Flk-1. CD45 was identied on 46.4% 0.9% of not-AT cells (Figure 1D). Freshly isolated AT cells were positive for Thy-1 (67.1% 3.7%; Figure 1F), CD31 (65.6% 3.2%; Figure 1G), and Flk-1 (56.9% 2.6%; Figure 1H), whereas they were negative for CD45 (2.3% 2.1%; Figure 1E).

Distribution of Transplantation EPCs in Cirrhotic Livers

In vivo FISH analysis to detect the Y chromosome (Figure 2, red) showed that transplantation living male EPCs could be detected in CCl4-induced cirrhotic female liver (Figure 2A). The male cirrhotic rat liver tissues were used as a positive control (Figure 2B), and the female cirrhotic rat liver tissues were used as a negative control (data not shown). Importantly, these EPCs labeled with red uorescence were observed adhering to the vasolateral surface of CD31 blood vessels (12 hours after EPC transplantation; Figure 2C), within vessel walls (1 day after EPC transplantation; Figure 2D), and near the vessels within the brotic areas (3 days after EPC transplantation; Figure 2E) of CCl4-treated livers. Most of the EPCs were observed around the portal tracts and brous septa from 1 to 4 weeks after EPC transplantation (1 week after EPC transplantation; Figure 2F). Additionally, after 4 sequential EPC transplantations, the transplantation EPCs labeled with red uorescence were located in brotic areas (Figure 2H and I). These EPCs were positive for CD31 (Figure 2J), whereas they were negative for -SMA (Figure 2J), cytokeratin-19 (Figure 2J) and albumin (data not shown) at 4 weeks after EPC transplantation. Similarly, 3 weeks after EPC transplantation, transplantation EPCs were observed in brotic areas of TAA-treated livers (data not shown). We did not observe the incorporation of EPCs into normal livers. Finally, we did not observe any inltrated cells with red uorescence in saline-infused livers treated with CCl4 or TAA treatment (data not shown).

Antibrogenic Effects of Transplantation EPCs in Cirrhotic Livers

Reduction of liver brosis 4 weeks following transplantations of EPCs was demonstrated by AzanMallory histologic staining (Figure 3A) and by immu-

nohistochemical analysis for type I collagen and bronectin (Figure 4A) in CCl4-treated livers. Livers treated with multiple EPC transplantation had fewer -SMA-positive and TGF- producing cells than did livers receiving saline and irradiated EPC treatment (Figure 4A). These inhibitory effects were observed ubiquitously throughout the liver. There were no signicant differences between saline- and irradiated EPC-treated livers. Semiquantitative analysis indicated that the relative extent of the brotic area was 5.3% 1.0% in livers that received multiple EPC transplantation compared with 10.9% 1.7% in livers treated with saline infusion and 10.3% 1.5% in livers treated with irradiated EPC transplantation in the CCl4-induced liver cirrhotic model (Figure 3C). Moreover, we compared the difference in the inhibitory effects on liver brosis between single and multiple EPC transplantations. Single EPC-transplantation livers (7.0% 1.1%) showed signicantly reduced liver brosis compared with livers receiving saline and irradiated EPC treatment (Figures 3A and C). More signicant, the brotic areas of multiple EPC-transplantation livers were signicantly less extensive than those of single EPC-transplantation livers (Figure 3C). A similar trend was noted in the TAA-induced liver cirrhotic model; the percentage of the brotic area in livers that received EPC transplantation, measured 3 weeks following multiple EPC transplantations (4.6% 2.0%), was signicantly lower than that in livers treated with saline infusions for 3 weeks (9.1% 2.9%) (Figures 3B and D). Realtime PCR showed that multiple EPC transplantations signicantly decreased the expression of hepatic mRNA for -2-(I)-procollagen, bronectin, -SMA, and TGF- compared with levels measured in salineinfused rats in the CCl4-induced liver cirrhotic model (Figure 4BE). Moreover, serum type IV collagen concentrations in multiple EPC-transplantation rats (17.1 6.4 ng/mL) were signicantly lower than in salineinfused liver rats (50.7 13.1 ng/mL) after 10 weeks of treatment with CCl4 (Table 2). Similarly, after 9 weeks of treatment with TAA, the serum type IV collagen concentrations in multiple EPC-transplantation rats (8.4 4.0 ng/mL) were also signicantly lower than in saline-infused liver rats (17.4 4.7 ng/mL; Table 3).

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Table 3. Serum Hepatic Enzyme and Type IV Collagen in TAA-Induced Liver

Type IV collagen (ng/mL) Intact TAA 6W TAA 9W TAA 9W saline multiple EPC 6.1 17.4 8.4 1.2 4.7 4.0a AST (U/mL) 64 135.2 207.1 189.5 18.6 16.1 19.2 29.7 ALT (U/mL) 32 63.4 79.5 65.8 5.6 6.5 20.2 12.7 Total bilirubin (mg/dL) 0.07 0.09 0.18 0.11 0.06 0.01 0.03 0.01a Total protein (g/dL) 5.9 5.36 5.30 5.88 0.27 0.31 0.18 0.11a Albumin (g/dL) 2.87 2.2 2.17 2.39 0.11 0.07 0.07 0.04a

W, weeks. aP .05 vs TAA 9W

saline.

Elevation of MMP Activity and Reduction of Tissue Inhibitor of Metalloproteinase-1 by Transplantation EPCs in CCl4-Treated Livers
As described above, we found that established liver brosis could be reduced by EPC transplantation. We speculated that some MMPs might be involved in this regression process. Real-time PCR demonstrated that multiple EPC-transplantation livers showed a signicant increase in MMP-9 mRNA expression 1 week after initial EPC transplantation, and this increase was persistently noted over the ensuing 2 weeks, compared with saline-infused livers in CCl4-induced cirrhotic liver (Supplementary Figure 1B; see supplementary material online at www.gastrojournal.org). MMP-2 and -13 mRNA expression in multiple EPC-transplantation livers was only signicantly up-regulated 1 week following the initial EPC transplantation, compared with levels in saline-infused CCl4-induced cirrhotic liver (Supplementary Figures 1A and C). We also analyzed the MMP-2 and -9 activities using gelatin zymography, as well as MMP-13 activity using Western blotting. Gelatin zymography showed that no bands for MMP-2 or -9 were detectable in normal liver. In contrast, after 6 weeks of CCl4 treatment, both pro- and active MMP-2 and -9 were detected (Figure 5A). Furthermore, the expression levels of pro- and active MMP-2 and pro- and active MMP-9 in multiple EPC-transplantation livers were signicantly stronger than those in saline-infused livers after 7 weeks of CCl4 treatment (Figures 5A and Supplementary Figures 1F and G). EPC lysates showed that EPC itself produced pro- and active forms of MMP-2 and -9 (Figure 5A). Film in situ zymography revealed that the MMP-2 and -9 activities

were greatly elevated in multiple EPC-transplantation livers compared with saline-infused livers and were localized in the brotic areas in which transplantation EPCs were incorporated after 7- and 10-week CCl4 treatment (Figure 5B). These gelatinolytic activities were completely blocked by the addition of 1, 10phenanthroline, an MMP inhibitor (Supplementary Figure 1H). Western blotting showed that only faint bands for both pro- and active MMP-13 were detectable in normal liver; however, following a 6-week CCl4 treatment, active MMP-13 was elevated (Figure 5C). The expression of active MMP-13 in multiple EPCtransplantation livers was signicantly stronger than that in saline-infused livers after 7 weeks of CCl4 treatment (Supplementary Figure 1I). EPC lysates showed that EPC itself produced active forms of MMP-13 (Figure 5C). In addition, an immunouorescence study showed that cells producing MMP-2, -9, and -13 included not only the transplantation EPCs but also the indigenous cells in vivo after 7 weeks of CCl4 treatment (Figure 5D). More of these indigenous MMP-2, -9, and -13 producing cells were detected in EPC-transplantation livers than in saline-treated livers (data not shown). We also measured the expression of tissue inhibitor of metalloproteinase (TIMPs) by real-time PCR. The expression level of TIMP-1 mRNA in multiple EPC-transplantation livers decreased and became signicantly lower after 10 weeks of CCl4 treatment compared with the saline-infused livers (Supplementary Figure 1D). In contrast, there was no signicant difference in TIMP-2 mRNA expression between multiple EPC-transplantation

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Figure 5. (A) Gelatin zymography indicated that both pro- and active-MMP-2 and -9 were increased in EPC-transplantation livers compared with saline-infused livers after CCl4 treatment for 7 weeks. EPC lysates showed that EPC itself produced active forms of MMP-2 and -9. (B) Film in situ zymography revealed that the gelatinolytic activities were greatly elevated in EPC-transplantation livers compared with saline-infused livers after 7 and 10 weeks of CCl4 treatment. Original magnication, 40 . (C) MMP-13 expression in livers after 7 weeks of CCl4 treatment. Western blotting showed that active-MMP-13 was increased in EPC-transplantation livers compared with saline-infused livers after 7 weeks of CCl4 treatment. EPC lysates showed that EPC itself produced active forms of MMP-13. Intact, normal livers; CCl4 6W, livers after 6 weeks of CCl4 treatment; CCl4 7W Multiple EPC, EPC-transplantation livers after 7 weeks of CCl4 treatment; CCl4 7W Saline, saline-infusion livers after 7 weeks of CCl4 treatment. (D) Immunouorescence showed that both PKH26 EPCs (red) and indigenous cells produced MMP-2, -9, and -13 (green) after 7 weeks of CCl4 treatment. Arrows indicate PKH26 EPCs (red) coexpressed MMP-2, -9, and -13 (green); arrowheads indicate MMP-2, -9, and -13 expressing PKH26- cells expressing MMP-2, -9, and -13. Tissues were stained with TO-PRO-3 iodide to visualize the nuclei (blue). Scale bar, 20 m for upper panel, 10 m for lower panel.

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Figure 6. (A) Immunohistochemical analysis of hepatocyte proliferation. Ki-67-positive hepatocytes were far more numerous in EPC-transplantation livers than in saline-infused livers after 10 weeks of CCl4 administration. Livers after 6 weeks of CCl4 treatment are shown on the left, saline-infusion livers after 10 weeks of CCl4 treatment are shown in the middle, and EPC-transplantation livers after 10 weeks of CCl4 treatment are shown on the right. Original magnication, 200. (B) The time course of the Ki-67 labeling index for hepatocytes in liver tissues. The Ki-67 labeling index for hepatocytes in livers of rats receiving EPC transplantation (light gray shaded columns) was signicantly higher than in livers of rats infused with saline (dark gray shaded columns) after 8 10 weeks of CCl4 treatment (*P .05, P .01, n 10). (C) The time course of the liver/body weight ratios. The liver/body weight ratios for rats receiving EPC transplantation (light gray shaded columns) were signicantly higher than those for rats infused with saline (dark gray shaded columns) after 10 weeks of CCl4 treatment (*P .05, n 10). KI-LI; Ki-67 labeling index; Intact, normal livers; CCl4 6W, livers after 6 weeks of CCl4 treatment; CCl4 10W Saline, saline-infused livers after 10 weeks of CCl4 treatment; CCl4 10W Multiple EPC, livers undergoing transplantation 4 times with EPC after 10 weeks of CCl4 treatment.

and saline-infused livers throughout the experiment (Supplementary Figure 1E).

Promotion of Hepatic Regeneration by Transplantation EPCs

To evaluate whether multiple EPC transplantations enhance the proliferation of hepatocytes in cirrhotic livers, we performed immunohistochemistry with the Ki-67 antibody (Figure 6A). In multiple EPC-transplantation livers, the percentage of Ki-67-positive hepatocytes increased progressively but insignicantly until week 7, but this percentage increased from 8 weeks (7.4% 1.5% vs 4.0% 0.6%, respectively) to 10 weeks (10.9% 2.2% vs 3.8% 0.9%, respectively) after CCl4 treatment, compared with saline-infused livers (Figure 6B). The ratio of the liver/body weight for rats receiving multiple EPC transplantations was signicantly higher than that for rats infused with saline after 10 weeks of CCl4 treatment (0.043 0.005 vs 0.035 0.006, respectively, Figure 6C). Next, we checked the mRNA expression levels of various growth factors known to be associated with hepatic regeneration using real-time PCR. Multiple EPC-transplantation livers showed a signicant increase in hepatocyte growth factor (HGF) mRNA expression 1 week after the initial EPC transplantation, and this increase was persistently noted over 10 weeks of CCl4 treatment, compared with saline-infused livers (Figure 7A). In multiple EPC-

transplantation livers, the expression of TGF- and epidermal growth factor (EGF) mRNA increased progressively but insignicantly until week 7 but increased from 8 to 10 weeks after CCl4 treatment, compared with salineinfused livers (Figure 7B and C). A similar trend was noted for vascular endothelial growth factor (VEGF) mRNA expression, although the increase was somewhat delayed compared with other regeneration factors. VEGF mRNA expression tended to increase at week 7, although insignicantly, but increased signicantly at week 10 after CCl4 treatment in multiple EPC-transplantation livers compared with saline-infused livers (Figure 7D). We also examined the expression of several growth factors in EPCs compared with RAECs. Both EPCs and RAECs were found by real-time PCR to express HGF, TGF- , EGF, and VEGF. Expression of HGF, TGF- , and EGF mRNA in EPCs was signicantly greater than in RAECs, whereas a signicant difference was not found for VEGF mRNA (Figure 7E). Therefore, serum concentrations of total protein and albumin were signicantly increased and alanine aminotransferase (ALT) and total bilirubin were signicantly decreased in multiple EPC-transplantation rats compared with saline-infused rats in the CCl4-induced cirrhotic model (Table 2). In the TAA-induced cirrhotic model, serum concentrations of total protein and albumin were also signicantly increased and total

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Figure 7. The expression of various growth factors in liver tissues and in EPCs. (AD) Quantitative real-time PCR analysis of mRNA expression levels of HGF, TGF- , EGF, and VEGF after treatment in EPC-transplantation (light gray shaded columns) and saline-infused (dark gray shaded columns) liver tissues. Expression of HGF, TGF- , EGF, and VEGF mRNA after CCl4 treatment for 10 weeks was signicantly greater in EPC-transplantation livers than in saline-infused livers. Results are expressed in arbitrary units (*P .05 compared with saline-infused livers per time point; n 10). Solid columns show livers after 6 weeks of CCl4 treatment. Intact, normal livers (open columns). (E) Quantitative real-time PCR analysis of mRNA encoding HGF, TGF- , EGF, and VEGF in EPCs (solid columns) and RAECs (open columns). Expression of HGF, TGF- , and EGF mRNA in EPCs was signicantly greater than in RAECs, whereas a signicant difference was not found for VEGF mRNA. Results are expressed in arbitrary units (n 5). *P .05, P .01; n.s., not statistically signicant.

bilirubin was also signicantly decreased in multiple EPC-transplantation rats compared with saline-infused rats (Table 3).

Up-Regulation of Survival Rate by Transplantation EPCs in CCl4-Treated Livers

The survival rates of single and multiple EPCtransplantation rats and saline-infused rats in the CCl4induced cirrhotic model were compared. The survival rate of multiple EPC-transplantation rats (70%) after 10 weeks of CCl4 treatment was signicantly higher than that of saline-infused rats (46%); however, no signicant difference was noted between single EPC-transplantation rats (60%) and saline-infused rats (Figure 8).

Discussion
Recent advances in stem cell research have revealed that BM cells, including hematopoietic stem cells, can differentiate into cells of other lineages that compose various tissues.511 In addition, the liver can support hematopoiesis in some situations.28 In our previous studies, we showed that EPC transplantation signicantly

Figure 8. Survival rate of CCl4 cirrhotic liver rat models. Signicantly prolonged animal survival was observed in the multiple EPC-transplantation groups. *P .05 compared with saline-treated groups; n.s., not statistically signicant.

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enhanced vascularization and improved survival rates after acute liver injury in mice.20 In the present study, we examined whether EPCs could inhibit brogenesis in vivo and whether EPC transplantation could be effective against established liver brosis. Clinically, this is an important issue. In fact, we showed that established liver brosis was eliminated and that liver function was also improved by EPC transplantation despite continuous administration of CCl4 or TAA. Moreover, proliferation of hepatocytes was increased approximately 3-fold (in comparison with controls). These ndings show that EPC transplantation could universally improve cirrhosis irrespective of the cause of liver damage. There is as yet no exclusive marker for EPCs.29 FACS analyses revealed that AT cells at day 7 of culture expressed Thy-1, CD31, and Flk-1 (Figure 1FH), all of which are markers for endothelial lineage.14,30 Approximately 40% of the AT cells were negative for Flk-1 expression at day 7 of culture. It has been reported that Flk-1 mRNA is initially expressed at low levels in human EPCs and that its expression gradually increases during culture for up to 21 days.31 Based on these characteristics of the AT cells, we dened the AT cells as the population of rat EPCs in the present study. Cirrhosis is associated with major alterations in both the quantity and composition of the extracellular matrix (ECM).1,2,32 In advanced disease stages, the liver contains abnormal amounts of ECM. Accumulation of ECM results from both increased protein synthesis and decreased degradation. The activity of MMPs that degrade the ECM is suppressed primarily by overexpression of TIMPs.33 Increased production of ECM is mediated primarily by activated hepatic stellate cells (HSCs). Activated HSCs are the most abundant ECM-producing cells and are a major source of TGF- production in the injured liver.1,2,32 In the current study, we found that EPC transplantation halts established liver brosis because of the suppression of -SMA-positive and TGF- producing activated HSCs (Figures 3 and 4). -SMA is one of the well-known markers for activated HSCs.1,2,32 We observed that the expression of -SMA, TGF- , and TIMP-1 were also suppressed by EPC transplantation (Figure 4D and 4E and Supplementary Figure 1D). It has been reported that TGF- inhibits apoptosis of activated HSCs induced by CD95 agonistic antibodies in vitro34 and that apoptosis of HSCs increases during the spontaneous regression of CCl4-induced liver brosis.35 This evidence indicated that brosis was likely to be reduced in the EPC-transplantation livers. This reduction, in turn, would likely be due to elimination of activated HSCs through apoptosis resulting from suppression of TGF- . There remains the possibility that activated HSCs might be converted to quiescent HSCs. However, no such conversion has yet been demonstrated. Increased expression of TIMP-1 has been shown in the development of rat liver brosis, and it plays an important role in liver brogenesis.36,37 Fur-

thermore, TIMP-1 exerts antiapoptotic activity on activated HSCs.38 40 It has been reported that the expression level of TIMP-1 mRNA decreases rapidly during the rst 4 weeks after the cessation of CCl4 treatment, and, consequently, the decreased expression of TIMP-1 contributes to the reduced numbers of activated HSCs.35 EPC transplantation reduces the expression of TGF- and TIMP-1 (Figure 4E and Supplementary Figure 1D) and may up-regulate the apoptosis of activated HSCs. This indicates that the reduction of activated HSCs contributes to the antibrotic effect of EPC transplantation. Increased collagenolytic activity is a major mechanism of brolysis.33,41 Fibrillar collagens are degraded by MMP-13 in rodents.42 During brolysis, MMP activity increases because of a rapid decrease of TIMP-1 expression. In addition, MMP-2 and MMP-9 activities were found to be progressively increased in rat liver brosis.43,44 In the present study, we reveal the following ndings: (1) After 6 weeks of CCl4 treatment, the MMP-2, -9, and -13 activities were increased in the brotic liver, (2) MMP-2, -9, and -13 activities were greatly elevated after EPC transplantation, and (3) EPC itself produced active forms of MMP-2, -9, and -13 (Figure 5). In rats, MMP-13 gene expression has been found to increase during the regression phase of CCl4-induced liver brosis.45 Similarly, in our study, the expression level of MMP-13 mRNA increased signicantly after 7 weeks of CCl4 treatment in EPC-transplantation livers compared with saline-infused liver (Supplementary Figure 1C). Furthermore, we also demonstrated by in situ zymography that active MMP-2 and -9 were located primarily in the brotic areas in which transplantation EPCs were incorporated (Figure 5B). Sakaida et al have demonstrated increased gelatinolytic activity in liver tissue after BM cell infusion.46 In addition, there is a very recent publication by Higashiyama et al demonstrating that MMP-9 and MMP-13 were produced by both BM-derived cells and indigenous cells.47 Similarly, our own study revealed that not only the transplantation EPCs but also the indigenous cells produced MMP-2, -9, and -13 and that the number of such MMPs-expressing indigenous cells increased after EPC transplantation (Figure 5D). These data all indicate that MMPs are involved in critical mechanisms underlying the EPC transplantation-induced brolysis. The activated MMPs are inhibited by a family of TIMPs.33,36 It has been reported that TIMP-1 gene expression is increased in animal and human liver brosis.36,37 Yoshiji et al showed that, once CCl4 was given, liver brosis was promoted in TIMP-1-transgenic mice and that the spontaneous resolution of brosis was attenuated.48,49 In EPC-transplantation livers, the expression level of TIMP-1 mRNA decreased signicantly after 10 weeks of CCl4 treatment compared with the saline-infused livers (Supplementary Figure 1D). These data suggest that active MMP-2, -9, and -13 of EPCs, along with

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reduced expression of TIMP-1, up-regulate active MMP-2, -9, and -13 levels in brotic liver, inducing enhanced brolysis in the EPC-transplantation liver. Increased expression levels of hepatic regeneration factors including HGF, TGF- , EGF, and VEGF, and enhanced proliferation of hepatocytes, were detected in EPC-transplantation livers (Figures 6 and 7). In our previous studies, we proved that transplantation human EPCs contributed to hepatic regeneration in mice by participating in neovascularization and expression of multiple growth factors such as HGF, HB-EGF, TGF- , and VEGF.20 Moreover, we have observed that transplantation of human EPCs, which express human VEGF and HGF, induces up-regulation of endogenous VEGF and HGF expression in acutely injured livers in mice.20 Furthermore, BM-derived rat EPCs expressed several growth factors such as HGF, TGF- , and EGF, which are wellknown to promote hepatocyte proliferation.50 These results, as well as our present ndings, suggest that incorporated EPCs promote hepatic regeneration not only by supplying various growth factors themselves but also by up-regulating the transcription of endogenous growth factors in the brotic liver. It has been reported that loss of TIMP-1 function accelerated the hepatocyte cell cycle via HGF activation.51 However, we could not clarify which plays the most important role in regenerating hepatocytes and in interactions of growth factors: increased expression of HGF, TGF- , EGF, and VEGF, or reduction of TGF- , which suppresses proliferation26 and induces apoptosis of hepatocytes.52 These questions are now under investigation in our laboratory. Several recent studies have demonstrated that BM and BM-derived cells, including EPCs, can repair injured heart, liver, and lung by reducing collagen deposition and remodeling.20,46,50,5356 Accordingly, we hypothesized that EPC transplantation could accelerate regeneration of the liver and reduce liver brogenesis. EPC transplantation was shown to induce several growth factors that can promote hepatic regeneration or ECM degradation and/or that can suppress ECM production. Moreover, we want to emphasize recent important ndings showing that liver organogenesis in fetal life is promoted by endothelial cells before they begin to function as vessels, and this process is independent of HGF.57 Additionally, it has been shown that BM-derived EPCs are major contributors to the regeneration of endothelium in mice after partial hepatectomy.58 These phenomena might be explained by our data. In conclusion, we found that transplantation of BMderived EPCs markedly ameliorates established liver brosis, liver function, and the survival rate via promotion of brolysis and hepatic regeneration. Our ndings suggest that the potential clinical value of cell therapy against liver brosis/cirrhosis might be greater than previously thought.

Supplementary Data
Supplementary data associated with this article can be found, in the online version, at doi:10.1053/j. gastro.2007.03.110.
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Received July 3, 2006. Accepted March 22, 2007. Address requests for reprints to: Toru Nakamura, MD, PhD, Division of Gastroenterology, Department of Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka, 830-0011, Japan. e-mail: ntoru@med.kurume-u.ac.jp; fax: (81) 942-34-2623. All authors declare no conict of interest to disclose. Supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, as part of a project for establishing new high technology research centers and center of excellence. The authors thank Mari Hagiwara for technical assistance in preparation of tissue sections, Masako Shinkawa and Kazuyo Handa for excellent technical assistance, and Masahisa Tsuji (The Chromosome Science Lab. Ltd, Hokkaido, Japan) for analyzing rat Y chromosome by FISH.

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Supplementary Figure 1. (AE) Quantitative real-time PCR analysis of mRNA expression levels of MMPs and TIMPs after treatment for EPCtransplanted (light gray shaded columns) and saline-infused (dark gray shaded columns) liver tissues. Expression of MMP-9 mRNA after CCI4 treatment for 7 to 8 weeks was signicantly up-regulated in EPC-transplanted livers compared with saline-infused livers. Expression of MMP-2 and -13 mRNA after CCI4 treatment for 7 weeks was signicantly greater in EPC-transplanted livers than in saline-infused livers. Expression of TIMP-1 mRNA after CCI4 treatment for 10 weeks was signicantly reduced in EPC-transplanted livers compared with saline-infused livers. Results are expressed in arbitrary units (*P .05 compared with saline-infused livers per time point; n 10). Solid columns show livers after 6 weeks of CCI4 treatment. Intact, normal livers (open columns). (F, G) Semiquantitative analysis of relative MMP-2 and -9 activity (*P .05, n 10). (H) MMP-PT in situ Zymo-Film analysis showed that these gelatinolytic activities were completely blocked by the addition of 1, 10-phenanthroline, an MMP inhibitor. Original magnication: 100 . (I) Semiquantitative analysis of relative MMP-13 activity (*P .05, n 10).