Fibrosis in Small Syngeneic Rat Liver Grafts Because of Damaged Bone Marrow Stem Cells From Chronic Alcohol Consumption

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1 ORIGINAL ARTICLE Fibrosis in Small Syngeneic Rat Liver Grafts Because of Damaged Bone Marrow Stem Cells From Chronic Alcohol Consumption Masayuki Hisada, 1,2 Xiuying Zhang, 1,3 Yoshihiro Ota, 1,2 Andrew M. Cameron, 1 James Burdick, 1 Bin Gao, 4 George Melville Williams, 1 and Zhaoli Sun 1 1 Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD; 2 Department of Surgery, Tokyo Medical University, Tokyo, Japan; 3 Department of Pathology, Beijing Capital Medical University, Beijing, China; and 4 Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD A patient with liver failure due to chronic and acute alcohol abuse under consideration for an urgent liver transplant shortly after stopping alcohol may have residual abnormalities that threaten transplant success, particularly for a small graft. To address this, we studied a model in which reduced-size (50%) Lewis rat livers are transplanted into green fluorescence protein transgenic Lewis recipients after they are fed alcohol or a control diet for 5 weeks. Here we show that normal small Lewis grafts transplanted to alcohol-fed Lewis hosts developed fibrosis, whereas no fibrosis was observed in control-fed recipients. Host-derived CD hydroxy-2 0 -deoxyguanosine (8-OHdG) cells were significantly increased in livers recovered from both alcohol-fed and control recipients, but only alcohol-fed recipients demonstrated co-staining (a marker of oxidative DNA damage). a smooth muscle actin (a-sma) staining, a marker for myofibroblasts, also co-localized with CD133 1 cells only in the livers of alcohol-fed recipients. Immunostaining and polymerase chain reaction analysis confirmed that chronic alcohol consumption decreased the proportion of bone marrow stem cells (BMSCs) expressing CD133, c-kit, and chemokine (C-X- C motif) receptor 4 markers and caused oxidative mitochondria DNA (mtdna) damage. Culture of CD133 1 cells from normal rats with medium containing 3% ethanol for 48 hours resulted in elevated mitochondrial 8-OHdG and mtdna deletion, and ethanol exposure diminished CD133 expression but dramatically increased a-sma expression. In conclusion, oxidative mtdna damage and deletions occur in BMSCs of chronic alcohol-fed recipients, and these damaged cells mobilize to the small liver grafts and become myofibroblasts where they play a key role in the subsequent development of fibrosis. Liver Transplantation AASLD. Received March 23, 2017; accepted July 7, Severe alcoholic hepatitis (SAH) is acute-on-chronic liver failure due to chronic and active severe alcohol abuse. These patients have a high mortality because effective therapies are lacking, so liver transplantation (LT) has become a lifesaving therapy. Normally, transplant centers require abstinence, usually for 6 months, Abbreviations: 8-OHdG, 8-hydroxy-2 0 -deoxyguanosine; a-sma, a smooth muscle actin; ALD, alcoholic liver disease; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BM, bone marrow; BMSC, bone marrow stem cell; bp, base pair; BSA, bovine serum albumin; CXCR4, chemokine (C-X-C motif) receptor 4; DAPI, 4 0,6-diamidino-2-phenylindole; DMEM, Dulbecco s Modified Eagle s Medium; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; GFP, green fluorescence protein; HSC, hepatic stellate cell; IgG, immunoglobulin G; LIN, lineage negative; LT, liver transplantation; mtdna, mitochondria DNA; NIH, National Institutes of Health; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PE, phycoerythrin; SAH, severe alcoholic hepatitis; SEM, standard error of the mean ORIGINAL ARTICLE before LT for patients with alcoholic liver disease (ALD), attempting to lower the risk of recidivism after LT and improve recovery from the transplant. However, the 6-month rule often cannot be met due to the urgency of most patients with SAH who are null responders to steroids after recent acute heavy alcohol use. Clinical trials in both Europe and the United States demonstrated that early LT is effective in patients with SAH who failed steroid treatment. (1-3) Rates of alcohol relapse were not different among study cohorts, although severe alcohol abuse may have been higher in the SAH group. (3) These promising results raised a question whether a living donor is suitable for SAH patients without a period of 6-month abstinence who have a statistically low priority for cadaveric organ transplantation based on the allocation system. Given these results, in addition to behavioral suitability any physiological impairment that will threaten a successful transplant must be addressed.

2 LIVER TRANSPLANTATION, Vol. 23, No. 12, 2017 Widespread application of cadaveric split or living donor LT has considerable potential to increase the pool of available organs and thus alleviate the problem of organ shortage. Although splitting of a cadaveric liver into 2 grafts for adult recipients can be performed successfully, sufficient function of undersized grafts is a major concern. (4,5) To minimize the risk for living donors, transplant surgeons aim to procure the least necessary liver volume, potentially leading to small grafts. An understanding of factors that impact regeneration of the small liver graft in recipients with a history of heavy alcohol use up to the time of LT is badly needed for expanding the donor organ pool for patients with SAH. Chronic alcohol consumption causes oxidative mitochondria DNA (mtdna) damage in hepatocytes (6) and inhibits liver regeneration after partial hepatectomy. (7,8) However, our previous publication (9) showed that a reduced-size (50%) liver from rats fed alcohol regenerated well when transplanted to a normal host. The effective regeneration was attributed to mobilization of host endogenous bone marrow stem cells (BMSCs). Recent studies (10) demonstrated that recruitment of bone marrow (BM) derived stem/progenitor cells to hepatic sinusoids after partial hepatectomy is required for normal liver regeneration. If Address reprint requests to Zhaoli Sun, M.D., Ph.D., Department of Surgery, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 771, Baltimore, MD Telephone: ; zlsun@jhmi.edu Masayuki Hisada conducted the majority of the experiments, analyzed data, and prepared the manuscript. Xiuying Zhang performed immunohistochemistry staining and polymerase chain reaction analyses. Yoshihiro Ota helped with animal models, histology, and fluorescenceactivated cell sorting analysis. George Melville Williams, Andrew M. Cameron, and Bin Gao provided suggestions for the project, and James Burdick edited the manuscript. Zhaoli Sun developed the concept, designed the overall study, supervised the project, and wrote the manuscript. Acknowledgment: This work was supported by the US NIH (UO1AA to Zhaoli Sun and Bin Gao). Additional supporting information may be found in the online version of this article. Copyright VC 2017 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /lt Potential conflict of interest: Nothing to report. damage to these BMSCs is also a prominent effect of chronic alcohol consumption, it is likely that this would impair liver regeneration. We hypothesized that a part of the systemic effect of chronic alcohol consumption is impairment of BMSCs due to oxidative damage to mtdna and that this damage persists in BMSCs after abstinence begins. This would result in damaged cells when mobilized to the injured liver that would fail to promote graft regeneration and moreover contribute to the development of fibrosis. To test this hypothesis, syngeneic reduced-size (50%) livers from normal Lewis rats were transplanted into alcohol-fed (5 weeks) green fluorescence protein (GFP) transgenic Lewis recipients. All recipients were then fed with normal food after transplantation. This model features 3 important factors which may bear on whether a living donor is suitable for alcoholic hepatitis patients without a 6-month abstinence: 1. Active alcohol abuse before transplantation. 2. The response of alcohol injured BMSCs to the regeneration of a small liver graft after a recent onset of abstinence. 3. Assessment of this possible mechanism for the threat to graft survival by the ability to distinguish the origin of cells in the liver via a genetic marker (GFP). Materials and Methods RAT STRAINS AND CARE Lewis (LEW, RT1 1 ) rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and used at 6-12 weeks of age. GFP transgenic Lewis rats were obtained from a National Institutes of Health (NIH) funded Rat Resource and Research Center, University of Missouri, Columbia, MO. All animals received humane care according to the criteria outlined in Guide for the Care and Use of Laboratory Animals (NIH publication 86-23, 1985 revision) and were under a protocol approved by the Johns Hopkins University Animal Care Committee. Wild-type or GFP Lewis rats, initially weighing 170 g, were fed a nutritionally adequate liquid diet containing 7% ethanol (Bioserv, Frenchtown, NJ) for 5 weeks, a period sufficient to develop alcoholic fatty liver. (9) LIVER TRANSPLANTATION Orthotopic whole or reduced-size (50%) small LTs from normal Lewis rats to alcohol-fed GFP transgenic Lewis rats were performed under isoflurane (Abbott ORIGINAL ARTICLE 1565

3 LIVER TRANSPLANTATION, December 2017 Laboratories, North Chicago, IL) inhalation anesthesia according to methods described previously. (11) The donor procedure for small LT consisted of removal of the left lateral lobe, the left portion of the middle lobe, and the anterior and posterior caudate lobes. This reduced the liver mass reproducibly by 50%. The liver grafts were weighed prior to transplantation to ensure that they were similar between alcohol-fed and nonalcohol-fed recipients ( g versus g; P ). The portal vein and infrahepatic vena cava were anastomosed using the cuff technique described before. (11) The common bile duct of the donor was cannulated with a small tube residing in the host bile duct and tied in place. The hepatic artery was not reconstructed. IMMUNOHISTOCHEMISTRY The 5-mm serially cut, frozen sections were fixed with acetone at ( 208C) for 10 minutes and dried for 1 hour at room temperature. The streptavidin-biotinperoxidase method with the DAKO Kit (Carpinteria, CA) was used to detect antigens. (12) After inactivation of endogenous peroxidase and blocking of nonspecific antibody binding, the specimens were incubated with antibodies specific to CD133 for undifferentiated stem cells (1:100, ab19898; Abcam, Cambridge, MA), Ki67 for dividing cells (1:100, ab16667; Abcam), or a smooth muscle actin (a-sma) for fibrosis precursors (1:200; Abcam) at 48C overnight. Subsequently, sections were incubated with biotin-conjugated donkey anti-rabbit (1:200, Jackson ImmunoResearch, West Grove, PA) secondary antibodies for 30 minutes at room temperature and then incubated with streptavidin-biotinperoxidase complex reagent (Vector Lab, Burlingame, CA) for 30 minutes at room temperature; 4,6-diamidino-2-phenylindole (DAPI) was used as the chromogen, and hematoxylin was used for counterstaining. MASSON S TRICHROME STAINING Liver tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at a thickness of 5 mm. The collagen deposition in liver tissue was evaluated by Masson s trichrome staining. IMMUNOFLUORESCENCE STAINING Frozen sections (5-mm) were fixed with acetone (2208C) for 10 minutes and dried for 1 hour at room temperature. Phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 5% normal donkey serum was used for blocking nonspecific background and dilution of antibodies. Sections were incubated for overnight at 48C with a mixture of a rabbit antibody to CD133 (1:100) and mouse antibody to a-sma or goat anti 8-OHdG (1:100; Japan Institute for the Control of Aging, Shizuoka, Japan) and then with Cy3 donkey anti-rabbit, fluorescein isothiocyanate (FITC) donkey anti-goat or donkey antimouse immunoglobulin G (IgG; 1:200; Jackson ImmunoResearch Inc.) for 1 hour at room temperature. Cell nuclei were stained blue with DAPI. Tissue sections were analyzed by confocal fluorescence microscopy. Double staining of CD133 and GFP was performed by immunofluorescent stains using frozen sections from GFP transgenic recipients. ISOLATION OF BM CELLS BM was extruded from tibia, humerus, and femur by flushing the cavity of the bones with PBS. (13) After flushing, the cells were filtered through a 70-mm filter to obtain a single cell suspension. Red blood cells were removed by using red blood cell lysing buffer (Sigma, Ronkonkoma, NY). BM cells were then resuspended in cold PBS containing 2% BSA. FLOW CYTOMETRY Single-cell suspensions ( ) of BM were analyzed for expression of lineage negative (Lin ) CD34, CD133, c-kit, and chemokine (C-X-C motif) receptor 4 (CXCR4) 1 stem cell markers, and for 8-OHdG, a stable and common oxidative DNA adduct. All antibodies used were from commercial sources: c-kit (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA), CXCR4 (1:100, ab2074; Abcam, Cambridge, MA), CD34 (1:100; R& D Systems, Minneapolis, MN), CD133 (1:100, ab19898; Abcam), anti 8-OHdG antibody (1:100; Japan Institute for the Control of Aging) and FITC anti-rabbit IgG, phycoerythrin (PE) anti-rabbit IgG, Day Light 647 anti- Goat IgG (1:200; Jackson ImmunoResearch Laboratories, Inc. West Grove, PA), and streptavidin-per CP (BD Pharmingen, San Jose, CA). Nonspecific antibody binding was blocked with donkey, mouse, and rat serum (Sigma) for 30 minutes. Cells were incubated with primary antibodies for 1 hour and then secondary antibodies for 45 minutes at 48C. The positive cells were counted by flow cytometry (fluorescence ORIGINAL ARTICLE

4 LIVER TRANSPLANTATION, Vol. 23, No. 12, 2017 activated cell sorting) using CELLQuest software (BD Biosciences). ENZYME-LINKED IMMUNOSORBENT ASSAY FOR MEASUREMENT OF 8-OHDG LEVELS IN mtdna IN BM CELLS The levels of 8-OHdG in mtdna were measured by enzyme-linked immunosorbent assay (ELISA) according to the methods described before. (6,12) Briefly, BM cells were homogenized in 5-mL of 50 mmol/l Tris- HCl (ph 7.4) with Dounce homogenizer. The homogenates were centrifuged at 800g for 10 minutes to precipitate the nuclear fraction. The supernatant was again centrifuged at 7000g for 10 minutes to yield the mitochondrial fraction. The mtdna was extracted using the DNeasy tissue kit (Qiagen, Santa Clara, CA) according to the manufacturer s instructions. The mtdna was digested to nucleotides, and the nucleotides were hydrolyzed to nucleosides. The nucleoside samples were used for the determination of 8-OHdG by a competitive ELISA kit (8-OHdG check; Japan Institute for the Control of Aging). DETECTION OF mtdna DELETION BY POLYMERASE CHAIN REACTION Total mtdna was extracted from mitochondria of BM cells as previously described. The primer sets for amplification of common mtdna deletion of 4834 base pair (bp), which were reported to be one of the most frequent deletions, (14,15) were 5-TTT CTT CCC AAA CCT TTC CT-3 ( bp) and 5- AAG CCT GCT AGG ATG CTT C-3 (13,108-13,126 bp). The primer sets for control amplification of wild-type mtdna were 5-GGT TCT TAC TTC AGG GGC CAT C-3 (15,782-15,892 bp) and 5-GTG GAA TTT TCT GAG GGT AGG C-3 (16,279-16,300 bp). Sequence and numbering are based on the rat complete mitochondrial genomes (GenBank accession number AJ ). BM CD133 1 CELL ISOLATION AND CULTURE BM CD1331 cells were isolated by immunomagnetic selection for CD133 using microbeads from the CD133 Cell Isolation Kit (Miltenyi Biotec, Cambridge, MA). For in vitro assays, isolated CD133 1 cells were inoculated in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 20 mmol/l 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 1 mmol/l ascorbic acid 2-phophate, and penicillin/ streptomycin at a density of cells/cm 2. CD cells were cultured with or without 65 mm of ethanol for 3 or 48 hours. Cells were then stained with a mitochondrial marker (MitoTracker Red CMXRos; Life Technologies, Carlsbad, CA) and anti 8-OHdG antibody. Double staining of CD133 and a-sma was also performed with immunofluorescent stains. STATISTICS Continuous variables were presented as the mean 6 standard deviation. Dichotomous variables were presented as both number and percentage values. Flow cytometry was analyzed using the Student t test (2-tailed), with dichotomous variables analyzed by the Fisher s exact test (2-tailed). P < 0.05 was considered significant. Results NORMAL SMALL LEWIS GRAFTS TRANSPLANTED TO ALCOHOL- FED LEWIS HOSTS SHOWED INHIBITED LIVER REGENERATION AND DEVELOPED FIBROSIS To determine the impact of chronic alcohol consumption on liver graft regeneration, wild type or GFP transgenic Lewis rats initially weighing 170 g were fed a nutritionally adequate liquid diet containing 7% ethanol for 5 weeks, a period sufficient to develop alcoholic fatty liver. Whole or reduced-size (50%) small livers from normal Lewis rat were transplanted into alcoholor control diet fed Lewis recipients. All control- and alcohol-fed Lewis recipients that received the syngeneic small or whole Lewis LTs survived greater than 4 weeks (n 5 8), although the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were higher in alcohol-fed compared with nonalcohol-fed recipients on day 3 after small LT (Fig. 1A). Immunohistochemistry staining demonstrated that Ki67 1 hepatocytes were increased in liver tissue sections recovered from small grafts on day 3 after transplantation (Fig. 1B,C). Interestingly, the number of Ki67 1 cells was significantly higher in ORIGINAL ARTICLE 1567

5 LIVER TRANSPLANTATION, December 2017 FIG. 1. Inhibited regeneration of syngeneic small liver grafts in alcohol-fed recipients. Reducedsized (50%) small livers were transplanted from normal Lewis rats to alcohol-fed (until the time of transplant) or nonalcohol-fed Lewis recipients. (A) Blood levels of AST and ALT at 3 days after transplantation. Values are the mean 6 SEM of 5 samples. *P < (B) Quantitative analysis of Ki67 1 cells in liver grafts at day 3 after transplantation. Values are mean 6 SEM of Ki67 1 cells per high-power field. **P < (C) Representative figures of immunohistochemistry staining. Ki67 1 cells show brown. Scale bar mm. Original magnification, control-fed recipients than that in alcohol-fed recipients (Fig. 1B). This was different from whole liver grafts, in which fewer Ki67 1 hepatocytes appeared in liver tissue sections recovered from either alcohol- or control-fed recipients on day 3 after transplantation (Supporting Fig. 1B). Using Masson s Trichrome stain to assess collagen levels in different stages of liver fibrosis, we found large amounts of fibrous septa and collagen bands in all alcohol-fed rats 14 days after small LT. No fibrosis was observed in control diet fed rat hosts (Fig. 2A) or hosts with whole LT (Supporting Fig. 1C). Immunohistochemistry staining revealed extensive a-sma 1 myofibroblast infiltration in the areas adjacent to fibrous bands in the livers of alcohol-fed rats (Fig.2B).Incontrast,fewa-SMA 1 cells were present in livers of control diet fed rats. Co-immunofluorescence staining of a-sma (red) and recipient marker GFP illustrates the participation of recipientderived myofibroblasts in fibrogenesis (Fig. 2D). These results suggest that chronic alcohol consumption inhibits regeneration of small liver grafts and failure to regenerate in alcohol-fed recipients may result in fibrosis. EXTRA-HEPATIC CD133 1 CELLS WERE SIGNIFICANTLY INCREASED IN SMALL LIVER GRAFTS RECOVERED FROM BOTH ALCOHOL-FED AND CONTROL RECIPIENTS, BUT ONLY ALCOHOL-FED RECIPIENTS DEMONSTRATED 8-OHDG CO-STAINING In other recent experiments, (16) we have shown that cells bearing the CD133 1 phenotype were important participants in liver regeneration. In order to identify oxidative DNA damage in BM-derived CD133 1 cells in the liver of alcohol-fed recipients, the expression of 8-OHdG, a stable and common oxidative DNA adduct, was analyzed by double staining with anti ORIGINAL ARTICLE

6 LIVER TRANSPLANTATION, Vol. 23, No. 12, 2017 FIG. 2. Histologic analysis of fibrosis in syngeneic small liver grafts in alcohol-fed recipients. Normal small Lewis liver grafts were transplanted to GFP 1 Lewis recipients previously fed alcohol until the time of transplant. Recipients were killed at 7 and 14 days after transplantation. (A) Collagen levels were evaluated in sections stained with Masson s Trichrome. Original magnification: (B) Immunohistochemistry staining for a-sma. a-sma 1 cells show brown. Original magnification: (C,D) Double staining for a-sma (red) and GFP (green) was performed by immunofluorescent stains using frozen sections. Almost all a-sma 1 cells co-stained with host marker GFP (yellow). Original magnification: CD133 and an anti 8-OHdG antibody. Indeed, CD133 1 cells were significantly increased in livers recovered from both alcohol-fed and control-fed recipients at 7 days after transplantation, but only alcoholfed recipients demonstrated 8-OHdG co-staining (green) in the majority of CD133 1 cells (Fig. 3A). Furthermore, immunofluorescence staining demonstrated expression of host GFP in nearly 100% of CD133 1 cells (Fig. 3B), indicating that CD OHdG 1 cells in the liver grafts derived from extrahepatic BM cells. a-sma staining, a marker for myofibroblasts, also co-localized with CD133 1 cells in the livers of only alcohol-fed recipients (Fig. 3C). Co-immunofluorescence staining (yellow) of a-sma (green) and BMSC marker CD133 (red) illustrates the participation of BM-derived stem cells in fibrogenesis. CHRONIC ALCOHOL CONSUMPTION DECREASED THE PROPORTION OF STEM CELLS WITH C-KIT, CD133, AND CXCR4 MARKERS To confirm whether damage to BMSCs is a systemic effect of alcohol consumption, BM cells were isolated from the thighbone in Lewis rats fed with 7% alcohol or a control diet for up to 4 weeks. Stem cells and progenitor cells have been phenotypically defined according to the expression of specific surface antigens such as CD34, c-kit, CD133, and CXCR4. They are termed Lin because they do not express lineage markers found in maturing blood cells. To further analyze the changes of stem/progenitor cells in BM after alcohol feeding, we used flow cytometry to analyze ORIGINAL ARTICLE 1569

7 LIVER TRANSPLANTATION, December 2017 FIG. 3. Histologic analysis of host CD133 1 stem cells in small liver grafts. (A) Double staining for CD133 and 8-OHdG in tissue sections from small liver grafts at 7 days after transplantation. Alcohol-fed recipients demonstrated 8-OHdG co-staining (green) in the majority of CD133 1 cells (red). (B) Immunostaining demonstrated expression of host GFP in CD133 1 cells (red). (C) Double staining for CD133 and a-sma in tissue sections from small liver grafts at 7 and 14 days after transplantation. a-sma staining, a marker for myofibroblasts, also co-localized with CD133 1 cells only in the livers of alcohol-fed recipients. Magnification: Lin BM cells according to the frequency of labeling with CD34, CD133, c-kit, and CXCR4 (Fig. 4A). The percentage of CD34 1 cells in the Lin stem cell enriched population remained the same in alcohol-fed rats compared with control diet fed rats (Fig. 4B). In contrast, CD1331, c-kit1, and CXCR4 1 cells in the Lin stem cell enriched population were significantly decreased in alcohol-fed rats compared with nonalcohol-fed rats. These results suggest that alcohol consumption damages BMSCs or inhibits development of these stem cell populations. CHRONIC ALCOHOL CONSUMPTION CAUSED OXIDATIVE mtdna DAMAGE IN BMSCS The expression of 8-OHdG was analyzed by double staining with anti 8-OHdG antibody plus other markers. Flow cytometry showed that the number of 8- OHdG 1 cells in the CD1331, c-kit1, andcd341 populations in BM dramatically increased in alcohol-fed rats compared with rats fed with control diets (Fig. 5A). To localize the expression of 8-OHdG in BMSCs, slides prepared via cytospin from single cell suspension of BM were stained with fluorescence labeled anti-8- OHdG antibody; 8-OHdG was clearly present in many BM cells after 4 weeks of ethanol feeding (Fig. 5B). However, few 8-OHdG 1 cells were recognized in rats fed with a control diet. The staining was localized mainly in the cytoplasm, indicating that this oxidative adduct was present in the mitochondrial and not nuclear DNA (the right panel of Fig. 5B). Quantitation of 8-OHdG in the mtdna of BM cells was done using a competitive ELISA method with the isolated mtdna (12) ; 8-OHdG was detected at low levels in the mtdna of BM cells recovered from rats fed with the control diet (Fig. 6A). The level of 8-OHdG in mtdna in BM cells recovered from rats fed with a diet containing 7% alcohol for 4 weeks was significantly higher than the baseline found in the rats fed with a control diet. To determine whether this injury resulted in mtdna deletions, template mtdna from BM cells after alcohol feeding was amplified using the primer pair between 7835 and 13,129 bp. Polymerase chain reaction (PCR) amplification (40 cycles; Fig. 6B) showed that increased mtdna deletions were found in BM cells at 2-4 weeks and reached the peak level at 3 weeks after ethanol consumption (Fig. 6C) ORIGINAL ARTICLE

8 LIVER TRANSPLANTATION, Vol. 23, No. 12, 2017 FIG. 4. Quantitative analysis of stem cells in BM of chronic alcohol-fed rats. BM cells were isolated from the thighbone in Lewis rats fed with 7% alcohol or control diets for 4 weeks. (A) Gates for cell sorting. Lin BM-derived stem cells were analyzed according to the intensity of labeling with CD34, CD133, c-kit, and CXCR4. (B) Cells sorted as in (A) that were from control- or alcohol-fed rats. The percentage of Lin CD1331, c-kit1, CXCR41, or triple positive (CD1331c-Kit1CXCR41)cells was significantly decreased in the BM in the animals fed with alcohol for 4 weeks, whereas the percentage of Lin CD34 1 cells remained the same. Quantitative data are represented as group means (bars; n 5 3). #P < 0.010, *P < ALCOHOL IS THE DIRECT CAUSE OF mtdna OXIDATION Two experiments were done to determine whether mitochondrial oxidation and myofibroblastic differentiation were direct or indirect effects of alcohol feeding. First, we sought another model of liver failure, common bile duct ligation, to find if the metabolic effects of liver failure itself caused stem cell failure. Two weeks after bile duct ligation in Lewis rats, small syngeneic LTs were done. ORIGINAL ARTICLE 1571

9 LIVER TRANSPLANTATION, December 2017 FIG. 5. Analysis of 8-OHdG expression in BMSCs in alcoholfed rats. (A) Flow cytometry. The expression of 8-OHdG in stem cells was analyzed by double staining with anti 8-OHdG antibody. The percentage of 8- OHdG 1 cells in the CD1331, c-kit1, and CD34 1 populations in BM dramatically increased in alcohol-fed rats compared with rats fed with control diets. Quantitative data are represented as group means (bars; n 5 3). *P < (B) Immunofluorescence staining. Slides prepared via cytospin from single cell suspension of BM were stained with fluorescence labeled anti 8-OHdG antibody (green). After 4 weeks of ethanol feeding, 8-OHdG was clearly present in BM cells and was localized mainly in the cytoplasm. Original magnification: Notably, there was no significantly increased mtdna damage in BMSCs (data not shown). All animals (7/7) survived and regenerated their livers without fibrosis at 14 days after transplantation. Second, the CD133 1 cells were cultured in Dulbecco s modified Eagle s medium in thepresenceofethanol(65mm)for48hours.mito tracker (red) and 8-OHdG (green) double staining demonstrated expression of 8-OHdG in most of the CD133 1 cells cultured with ethanol, but 8-OHdG was absent in cells cultured without ethanol (Fig. 7A). Interestingly, ethanol exposure diminished CD133 expression but dramatically increased a-sma expression (Fig. 7B). PCR analysis of mtdna demonstrated that common deletions occurred in CD133 1 cells at 48 hours after culture with 65 mm ethanol (Fig. 7C). These results support the position that alcohol directly causes mtdna oxidation, culminating in direct myofibroblastic differentiation and failure of stem cell facilitation of regeneration. Discussion Chronic alcohol abuse is directly toxic to hepatocytes, and it is generally acccepted that this is a major factor in fibrotic degeneration culminating in cirrhosis and liver failure. In addition, the current studies provide a new insight into the magnitude of liver injury caused secondarily by extrahepatic alcohol toxicity to the BM cells that are necessary for hepatic regeneration. This is demonstrated by our results in which normal but small GFP livers were transplanted using GFP1 recipients into an environment tainted by recent alcohol loading. Rather than being able to regenerate, the small livers became fibrotic, and the a-sma cells responsible for the fibrosis were derived from the GFP1 recipient. Moreover, when we repeated the identical transplants but used recipients suffering from chronic liver failure 1572 ORIGINAL ARTICLE

10 LIVER TRANSPLANTATION, Vol. 23, No. 12, 2017 FIG. 6. The mtdna damage and deletions in BM cells after ethanol feeding. (A) Levels of 8-OHdG in mtdna of BM cells from control diet fed and alcohol-fed Lewis rats for 4 weeks. Extracted mtdna from BM cells was enzymatically hydrolyzed into nucleosides, and levels of 8-OHdG were measured by an ELISA kit. Data represent mean 6 SEM of n 5 3 per group. *P < (B) PCR analysis of mtdna deletion in BM cells. Template mtdna of BM cells from control diet fed or alcoholfed rats was amplified in a conduction of 40 cycles using the primer pair between 7835 and 13,129 bp. The mtdna deletion was increased in rats fed with alcohol. (C) Semiquantitative analysis (common deletion/wild mtdna) showed that increased mtdna deletions were found in BM cells, up to the peak level at 3 weeks after ethanol consumption. Values are mean 6 SEM of 3 samples. *P < caused by bile duct ligation, all survived and there was no fibrosis in small liver grafts (unpublished data). Moreover, when we repeated the identical transplants but used recipients suffering from chronic liver failure caused by bile duct ligation, all survived and there was no fibrosis in small liver grafts (unpublished data). This rules out a role for the presence of liver failure before the transplant per se. Therefore, the alcoholrelated oxidative injury to critical stem cells prior to the transplant initiated the fibrotic changes present in the originally normal LT tissue. BMSCs bearing markers CD133, c-kit, and CD34 contained 3-4 times greater amounts of the DNA oxidative adduct 8-OHdG than controls at 3-4 weeks after alcohol feeding. Recipient GFP 1 CD133 1 cells were present in the transplant, they co-stained with 8-OHdG, and they co-localized with a-sma labeled cells. These cells were positioned in the liver at 2 weeks next to strands of fibrous tissue. Thus, the CD133 1 phenotypes were the ones most likely responsible for fibrotic changes. The damage was in mitochondrial DNA, not nuclear DNA, because mitochondrial DNA is more sensitive to oxidative stress and lacks a robust DNA repair mechanism. (14,15) In vitro studies were undertaken to further study the mechanims leading to fibrosis. Isolated CD133 cells were exposed to 3% alcohol for 2 days. As illustrated in Fig. 7, the oxidative adduct 8-OHdG was present in most of the mitochodria. At day 2, a-sma production was plentiful. Thus, alcohol is capable of directly causing oxidative injury to critical BM cells which initiate fibrosis when these cells are drawn to the normal small liver. No a-sma1, 8-OHdG1CD133 1 cells, or fibrosis was seen in control rats fed a normal diet. Interestingly, analysis of liver biopsies from a male patient who received a LT from a female donor and subsequently developed alcoholic hepatitis demonstrated that a significant proportion of the hepatic myofibroblasts were of recipient origin. (17) Our finding that recipient BMSCs may become myofibroblasts is not unique. There are now multiple reports in BM transplantation models suggesting that progenitor cells from the BM can migrate to the injured liver and differentiate into stellate cells and myofibroblasts. In the most quantitative study, Russo et al. carried out male to female BM transplants and found that 70% of fibrogenic myofibroblasts were derived from transplanted marrow cells. Transplantation of BM cells from mice with a mutant, degradation-resistant collagen I resulted in increased fibrosis. (18) A variety of other studies, most of which involved whole BM transplants, yielded similar evidence that marrow-derived progenitor cells could generate fibrogenic myofibroblasts. (19-21) A recent study showed that both BM-derived CD34 1 fibrocytes and hepatic stellate cells (HSCs) are involved in biliary fibrogenesis in Abcb4 / mice. (22) However, a study by Higashiyama et al. (23) found no evidence that any collagen expression in the injured liver was from bone marrow-derived cells. In an elegant review, Kallis and ORIGINAL ARTICLE 1573

11 LIVER TRANSPLANTATION, December 2017 FIG. 7. Effect of ethanol on oxidative mtdna damage and deletion in primary cultured BM CD133 1 cells. Naive cultured CD133 1 cells were incubated with 3% ethanol for 48 hours. (A) Double staining for Mito tracker (red) and 8- OHdG (green). Confocal microscopy demonstrated expression of 8-OHdG in most of the CD133 1 cells cultured with 3% ethanol, but 8-OHdG was absent in cells cultured without ethanol. The staining was predominantly colocalized to Mito trackers. (B) Immunofluorescence double staining for a-sma and CD133 in cultured CD133 1 cells. Ethanol exposure diminished CD133 expression but dramatically increased a-sma expression. (C) PCR analysis of mtdna deletion in cultured CD133 1 cells. The common deletions occurred in CD133 1 cells at 48 hours after culture with 3% ethanol. Data represent 4 independent experiments. Forbes (24) summarized the existing studies, suggesting that heterogeneity in the population of BM-derived stem cells explained these divergent results. Mesenchymal stem cells, which are resistant to radiation, and hematopoietic stem cells, which are radiosensitive, appear to have different abilities to repopulate the liver, and differences in experimental techniques may select for one versus the other. Although the majority of a-sma labeled graft cells were co-stained with recipient GFP in alcohol-fed rats with the small liver transplants, a few a-sma positive cells were GFP negative, indicating that donor 1574 ORIGINAL ARTICLE

12 LIVER TRANSPLANTATION, Vol. 23, No. 12, 2017 myofibroblasts are also likely to contribute to fibrosis. Three sources of myofibroblasts have been identified (25) : HSCs in hepatotoxic liver injury, portal fibroblasts in cholestatic liver injury, and fibrocytes in any inflamed liver. It is possible that these cells were activated due to increased injury and decreased regeneration of small liver grafts in alcohol-fed recipients and became myofibroblasts. Immunostaining and PCR analysis confirmed that oxidative mtdna damage and deletions occurred in BM cells of rats fed alcohol for 4 weeks, especially in Lin CD341, ckit1, and CD133 1 cells. The common deletions of mtdna in rats and humans are flanked by 2 homologous repeats that span a region that encodes 5 kinds of transfer RNA. Included in the omissions are respiratory enzyme subunits for complexes I, IV, and V. As a result, the injury to mtdna caused by chronic alcohol consumption is likely to be progressive. Cells surviving with impaired mitochondrial respiratory function are more likely to continue to suffer from faulty reduction of O 2, sustaining ever increasing oxidative stress, and are less likely to meet the metabolic needs of normal stem cell differentiation. In previous studies, we have shown that stem cells travelling from the BM can assist in liver regeneration after damage in the host. (9,16,26) The remarkable stem cell damage due to alcohol that we have demonstrated is likely to decrease regeneration and increase fibrosis and therefore be an important cause of chronic alcoholic liver damage. There are 2 important implications for LT in these studies that demonstrate functional BMSC damage that persists after alcohol intake has stopped. The first issue relates to the increased demand for livers leading to expanded criteria for donors. Outcomes with these organs vary from center to center, and much activity relates to finding markers which will predict function accurately. It may be important to find methods for assaying stem cell function in potential recipients because these livers from expanded criteria donors may require significant help from BMSCs. In the case of early LT for SAH without the 6-month period of abstinence, caution is indicated when living donor or split liver is considered. Failure of graft regeneration may result in small-for-size syndrome and/or fibrosis. Second, in the case of ALD a strict 6-month period of abstinence was enforced at first, but as results showed relative freedom from alcohol relapse and good acute outcomes, this requirement has lapsed. It will be important to monitor the outcomes in the patients transplanted after brief periods of abstinence when their stem cells may be still stricken. The impact of stem cell mtdna oxidation is likely to be less for whole organ grafts that will not need as much stem cell input for regeneration. 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