Transforming Growth Factor β Neutralization Ameliorates Pre-Existing Hepatic Fibrosis and Reduces Cholangiocarcinoma in Thioacetamide-Treated Rats.
ABSTRACT Considerable evidence has demonstrated that transforming growth factor β (TGF-β) plays a key role in hepatic fibrosis, the final common pathway for a variety of chronic liver diseases leading to liver insufficiency. Although a few studies have reported that blocking TGF-β with soluble receptors or siRNA can prevent the progression of hepatic fibrosis, as yet no evidence has been provided that TGF-β antagonism can improve pre-existing hepatic fibrosis. The aim of this study was to examine the effects of a murine neutralizing TGF-β monoclonal antibody (1D11), in a rat model of thioacetamide (TAA)-induced hepatic fibrosis. TAA administration for 8 weeks induced extensive hepatic fibrosis, whereupon 1D11 dosing was initiated and maintained for 8 additional weeks. Comparing the extent of fibrosis at two time points, pre- and post-1D11 dosing, we observed a profound regression of tissue injury and fibrosis upon treatment, as reflected by a reduction of collagen deposition to a level significantly less than that observed before 1D11 dosing. Hepatic TGF-β1 mRNA, tissue hydroxyproline, and plasminogen activator inhibitor 1 (PAI-1) levels were significantly elevated at the end of the 8 week TAA treatment. Vehicle and antibody control groups demonstrated progressive injury through 16 weeks, whereas those animals treated for 8 weeks with 1D11 showed striking improvement in histologic and molecular endpoints. During the course of tissue injury, TAA also induced cholangiocarcinomas. At the end of study, the number and area of cholangiocarcinomas were significantly diminished in rats receiving 1D11 as compared to control groups, presumably by the marked reduction of supporting fibrosis/stroma. The present study demonstrates that 1D11 can reverse pre-existing hepatic fibrosis induced by extended dosing of TAA. The regression of fibrosis was accompanied by a marked reduction in concomitantly developed cholangiocarcinomas. These data provide evidence that therapeutic dosing of a TGF-β antagonist can diminish and potentially reverse hepatic fibrosis and also reduce the number and size of attendant cholangiocarcinomas.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Immune response plays an important role in the development of hepatic fibrosis. In the present study, we investigated the effects of quercetin on hepatitis and hepatic fibrosis induced by immunological mechanism. In the acute hepatitis model, quercetin (2.5 mg/kg) was injected iv into mice 30 min after concanavalin A (Con A) challenge. Mice were sacrificed 4 or 24 h after Con A injection, and aminotransferase tests and histopathological sections were performed. Treatment with quercetin significantly decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Consistent with this observation, treatment with quercetin markedly attenuated the pathologic changes in the liver. A hepatic fibrosis model was also generated in mice by Con A challenge once a week for 6 consecutive weeks. Mice in the experimental group were treated with daily iv injections of quercetin (0.5 mg/kg). Histopathological analyses revealed that treatment with quercetin markedly decreased collagen deposition, pseudolobuli development, and hepatic stellate cells activation. We also examined the effects of quercetin on the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transforming growth factor beta (TGF-β) pathways by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). NF-κB and TGF-β production was decreased after treatment with quercetin, indicating that the antifibrotic effect of quercetin is associated with its ability to modulate NF-κB and TGF-β production. These results suggest that quercetin may be an effective therapeutic strategy in the treatment of patients with liver damage and fibrosis.Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 08/2014; 47(8):655-61. · 1.08 Impact Factor
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ABSTRACT: Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy with limited treatment options and low survival rates. Currently, there are no curative medical therapies for CCA. Recent advances have enhanced our understanding of the genetic basis of this disease, and elucidated therapeutically relevant targets. Therapeutic efforts in development are directed at several key pathways due to genetic aberrations including receptor tyrosine kinase pathways, mutant IDH enzymes, the PI3K-AKT-mTOR pathway, and chromatin remodeling networks. A highly desmoplastic, hypovascular stroma is characteristic of CCAs and recent work has highlighted the importance of targeting this pathway via stromal myofibroblast depletion. Future efforts should concentrate on combination therapies with action against the cancer cell and the surrounding tumor stroma. As the mutational landscape of CCA is being illuminated, molecular profiling of patient tumors will enable identification of specific mutations and the opportunity to offer directed, personalized treatment options.Seminars in Liver Disease 11/2014; 34(4):456-64. · 5.12 Impact Factor
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ABSTRACT: Mating shuts down a 2-methoxyestradiol (2ME)-dependent, non-genomic activity that is responsible for accelerating egg transport in the rat oviduct. The aims of this work were to investigate the role of TGFβ1 in this 2ME-reduced activity and to determine the effect of mating on the expression and distribution of TGFβ1 and its receptor TGFBR3 in the rat oviduct. We determined the level of TGFβ1 in the plasma and oviductal fluid at 1, 3, or 6 hr during Day 1 of the oestrous cycle in unmated or mated animals. We then examined if 2ME accelerates oviductal egg transport in unmated rats that were previously treated with a neutralizing TGFβ1 antibody. The expression of Tgfb1 and Tgfbr3 mRNA and the level and distribution of TGFBR3 protein in the oviduct were also determined at these time points. Mating decreased TGFβ1 in the plasma, but not in the oviductal fluid, whereas antibody neutralization of circulating TGFβ1 did not prevent the effect of 2ME on egg transport. Mating decreased Tgfb1 and hastened the increase in TGFBR3 abundance in the myosalpinx. These results indicate that mating decreased circulating levels of TGFβ1 without shutting down the non-genomic 2ME response that normally accelerates egg transport. Levels of Tgfb1 transcript and TGFBR3 protein, however, changed in the myosalpinx of mated rats, suggesting a role for mating-associated factors in the autocrine and paracrine effects of TGFβ in the oviduct. Mol. Reprod. Dev. 2014. © 2014 Wiley Periodicals, Inc.Molecular Reproduction and Development 10/2014; · 2.68 Impact Factor
Transforming Growth Factor b Neutralization
Ameliorates Pre-Existing Hepatic Fibrosis and Reduces
Cholangiocarcinoma in Thioacetamide-Treated Rats
Hong Ling*, Eric Roux, Donna Hempel, Jingzang Tao, Mandy Smith, Scott Lonning, Anna Zuk,
Cynthia Arbeeny, Steve Ledbetter
Tissue Protection and Repair, Sanofi-Genzyme R&D Center, Framingham, Massachusetts, United States of America
Considerable evidence has demonstrated that transforming growth factor b (TGF-b) plays a key role in hepatic fibrosis, the
final common pathway for a variety of chronic liver diseases leading to liver insufficiency. Although a few studies have
reported that blocking TGF-b with soluble receptors or siRNA can prevent the progression of hepatic fibrosis, as yet no
evidence has been provided that TGF-b antagonism can improve pre-existing hepatic fibrosis. The aim of this study was to
examine the effects of a murine neutralizing TGF-b monoclonal antibody (1D11), in a rat model of thioacetamide (TAA)-
induced hepatic fibrosis. TAA administration for 8 weeks induced extensive hepatic fibrosis, whereupon 1D11 dosing was
initiated and maintained for 8 additional weeks. Comparing the extent of fibrosis at two time points, pre- and post-1D11
dosing, we observed a profound regression of tissue injury and fibrosis upon treatment, as reflected by a reduction of
collagen deposition to a level significantly less than that observed before 1D11 dosing. Hepatic TGF-b1 mRNA, tissue
hydroxyproline, and plasminogen activator inhibitor 1 (PAI-1) levels were significantly elevated at the end of the 8 week TAA
treatment. Vehicle and antibody control groups demonstrated progressive injury through 16 weeks, whereas those animals
treated for 8 weeks with 1D11 showed striking improvement in histologic and molecular endpoints. During the course of
tissue injury, TAA also induced cholangiocarcinomas. At the end of study, the number and area of cholangiocarcinomas
were significantly diminished in rats receiving 1D11 as compared to control groups, presumably by the marked reduction of
supporting fibrosis/stroma. The present study demonstrates that 1D11 can reverse pre-existing hepatic fibrosis induced by
extended dosing of TAA. The regression of fibrosis was accompanied by a marked reduction in concomitantly developed
cholangiocarcinomas. These data provide evidence that therapeutic dosing of a TGF-b antagonist can diminish and
potentially reverse hepatic fibrosis and also reduce the number and size of attendant cholangiocarcinomas.
Citation: Ling H, Roux E, Hempel D, Tao J, Smith M, et al. (2013) Transforming Growth Factor b Neutralization Ameliorates Pre-Existing Hepatic Fibrosis and
Reduces Cholangiocarcinoma in Thioacetamide-Treated Rats. PLoS ONE 8(1): e54499. doi:10.1371/journal.pone.0054499
Editor: Johannes Haybaeck, Medical University Graz, Austria
Received August 7, 2012; Accepted December 12, 2012; Published January 17, 2013
Copyright: ? 2013 Ling et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no external funding related to this manuscript to report.
Competing Interests: The authors have read the journal’s policy and have the following conflict: The authors are employees of Sanofi-Genzyme R&D Center, a
Sanofi company. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: Hong.email@example.com
Liver cirrhosis is a common end consequence of a variety of
chronic liver diseases. Its underlying pathology, fibrosis, represents
the common response of the liver to toxic, infectious, or metabolic
agents [1–3]. Hepatic fibrosis, i.e., excess deposition of extracel-
lular matrix proteins, is traditionally viewed as an irreversible
pathological process involving multiple cellular and molecular
events [2,4–5]. In most patients with liver cirrhosis, disease
pathology increases in severity and does not regress, leading
ultimately to liver insufficiency and to the development of liver
carcinoma. However, recent evidence suggests that liver fibrosis is
dynamic and can be bidirectional, involving phases of progression
and regression , offering an opportunity for therapeutic
intervention to halt or reverse progression.
Transforming growth factor b (TGF-b) is a pleiotropic cytokine,
which regulates numerous essential cell functions. Considerable
evidence has accumulated showing that excess expression of TGF-
b induces and orchestrates intracellular signaling events leading to
increased matrix protein deposition and ultimately liver fibrosis
[7–9]. TGF-b1 is the main isoform mediating liver fibrosis through
autocrine and paracrine effects on various hepatic and infiltrating
cell types [7–9]. This pathological process also involves major
changes in the regulation of matrix degradation, in which
plasminogen activator inhibitor 1 (PAI-1), a downstream effector
of TGF-b signaling, may be a key player [10–11]. TGF-b
mediated changes to the structure and biophysical properties of
the extracellular micro-environment may also promote the
appearance and growth of neoplastic epithelial cells (16). However,
the role of TGF-b in this context is complex as this molecule also
promotes epithelial mesenchymal transdifferentiation (EMT), cell
invasiveness and metastasis [12–13], whereas in other settings
TGF-b functions as a tumor suppressor [14–15].
Given the prominent role of TGF-b in hepatic fibrosis, several
approaches to abrogate the effect of TGF-b have been reported.
These therapeutic strategies have been shown to be effective in
preventing liver fibrosis in several animal models. For example,
adenovirus-mediated local expression of dominant negative type II
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TGF-b receptor (TbRII) in liver and skeletal muscle significantly
reduced the extent of hepatic fibrosis in a thioacetamide (TAA)-
induced liver fibrosis model . Additionally, engineered forms
of soluble TGF-b receptor II, which act as a scavenger of this
cytokine, or RNA interference targeting TGF-b1, prevent
fibrogenesis in rodent models of liver disease [17–19]. These
studies have clearly established an anti-fibrotic role for TGF-b
antagonists in preventing liver fibrogenesis. However, the agents
were administered at the time of injury, at an early stage of disease
when substantial fibrosis was not yet developed, or in models that
could spontaneously regress after the toxic agents were removed.
Therefore, these studies do not address the therapeutic utility of
TGF-b antagonism in a setting of pre-existing hepatic fibrosis. The
aim of the present study was to investigate the effects of a TGF-b
neutralizing antibody, 1D11, in a rat model of TAA-induced
hepatic fibrosis, accompanied with the development of cholangio-
carcinoma (CCA) that recapitulates the histological features and
progression of human CCA [20–21]. The results suggest that
antagonizing TGF-b may reverse pre-existing hepatic fibrosis by
disrupting TGF-b synthesis, reducing extracellular matrix pro-
duction and promoting matrix degradation. Unexpectedly, this
therapeutic approach also substantially reduced TAA-induced
Materials and Methods
This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. All
protocols were approved by Genzyme’s Institutional Animal Care
and Use Committee (permit Number: 03-0918-2-BC).
A murine IgG1 monoclonal antibody, 1D11, which neutralizes
all three mammalian TGF-b isoforms (b1, b2 and b3), was
produced at Genzyme Corporation (Framingham, MA). This
antibody has a circulatory half-life of 5.5 days in rats when
administered by intraperitoneal injection. An isotype-matched
irrelevant murine IgG1 monoclonal antibody, 13C4, also
produced by Genzyme Corporation, was used as a control
Adult Fischer 344 rats (Charles River Laboratories, Worchester,
MA) weighing 280 grams (8–10 weeks old) were housed in an air-,
temperature-, and light-controlled environment. Based on our
pilot studies, the TAA-induced hepatic fibrosis model in Fischer
rat (F344) was chosen as this model has irreversible hepatic fibrosis
which is consistent among individual animals, and disease
pathology is comparable to humans. Experimental design and
dosing regimen are shown in Figure 1. For these studies, rats were
dosed with TAA intraperitoneally for 8 weeks (300 mg/kg three
times weekly for 6 weeks, followed by two times weekly for 2
weeks, Figure 1). A group of rats received vehicle buffer (phosphate
buffered saline, PBS) and served as normal controls. At the end of
8 weeks, TAA was withdrawn and six rats were sacrificed to
establish pre-existing hepatic fibrosis. The remaining animals were
divided into three groups and were given PBS, 13C4 or 1D11.
13C4 and 1D11 were dosed at 5 mg/kg, 3 times per week, IP. At
sacrifice, livers were perfused with sterile PBS. Sample collection
included harvesting the identical portion of the right lobe of the
liver to avoid lobe to lobe variability. Samples were then quickly
frozen on dry ice and stored at 280uC until use. Samples were
processed for the analysis of mRNA, measurement of hydroxy-
proline content, or Western blotting. Freshly harvested liver
samples were also fixed in 10% buffer neutralized formaldehyde
for 24 hours for histopathological examination, morphometric
analysis and immunostaining.
Blood Chemistry Analysis
Blood was drawn at week 0, 3 and 8 (with TAA dosing), and 11
and 14 weeks (3 and 6 weeks post treatments, respectively). Plasma
samples were analyzed for albumin and liver enzymes, alanine
aminotransferase (ALT), and aspartate aminotransferase (AST)
using a Roche Cobas Automate Biochemistry Analyzer (Roche
Diagnostics, Indianapolis, IN).
Paraffin embedded liver sections (5-mm) were stained with
hematoxylin-eosin (H&E) or picrosirius red. Assessment of the
histopathological changes was conducted by applying conventional
semi-quantitative scoring system for fibrosis, as described . All
evaluations were conducted blindly. Briefly, histopathological
changes were assessed based on biliary epithelial neoplasia, fibrous
Figure 1. Experimental protocol of TAA induced liver fibrosis. Fischer rats were dosed with TAA intraperitoneally for 8 weeks (300 mg/kg for
6 weeks, three times weekly, then two times weekly for 2 weeks). A cohort of rats received PBS as normal controls. At the end of 8 weeks, TAA was
withdrawn and rats were divided into three cohorts that were given PBS, 13C4 or 1D11 for 8 weeks. 13C4 and 1D11 were dosed at a concentration of
5 mg/kg, intraperitoneally, three times per week.
TGF-b Neutralization in Hepatic Fibrosis
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deposition, and fibrous bridging between portal areas or between
portal areas and central veins, with a greater attention to the
extent of collagen deposition. Semi-scores were given by estimat-
ing the lesions per section and the completion of fibrous bridging,
as follows: 0=none, 1=,25%, 2=25–50%, 3=,75% (with full
bridging, cirrhotic), and 4=.75% (severe cirrhotic), respectively.
Data are expressed as mean 6 SE.
Morphometric analysis was performed on liver sections stained
with picrosirius red for collagen deposition and cytokeratin (CK)
for neoplastic biliary cells, respectively. Stained slides were
scanned using ChromaVision automated imaging scanning system
(ChromaVision Medical Systems, San Juan Capistrano, CA), and
quantified with MetaMorph software (Molecular Device, Sunny-
vale, CA). Liver sections were scanned in a blinded manner, the
captured bright-field images were digitized and the area and
optical densities of the chromogen were quantified. To quantify
the area of cholangiocarcinoma consisting of neoplastic biliary
epithelium and fibrotic stromal matrix, the area of cholangiocar-
cinoma was measured manually with the MetaMorph program by
tracing every tumor outline.
Measurement of Liver Hydroxyproline Content
For the quantitation of total hepatic collagen, perfused livers
were excised, lyophilized and weighed. Lyophilized tissue was then
hydrolyzed in 2 N NaOH (final concentration: 100 mg dry tissue/
ml) at 120uC for 25 minutes. Ehrlich’s reagent (Sigma, St. Louis,
MO) was added to the hydrolysates for color reaction and
absorbance was measured at 550 nm. Hepatic hydroxyproline
content was expressed as ug hydroxyproline per mg of dry tissue.
Trans-4-hydroxy-L-proline (Sigma) was used as a standard.
RT-PCR Analysis of TGF-b gene Expression
Liver samples were placed in RNAlater (Ambion, Austin, TX)
immediately after harvesting and stored at 280uC until analysis.
Tissues were homogenized in 0.5 ml of Trizol reagent (Gibco,
Grand Island, NY) and total RNA was extracted and purified
using standard RNAeasy mini kit (Qiagen, Valencia CA). Specific
fluorogenic probes were designed for rat TGF-b1, TGF-b2, and
TGF-b3. Random hexamer primer was included in the reverse
transcription polymerase chain reaction (RT-PCR); cDNA was
generated from 2 mg of RNA by using BD Biosciences (Woburn,
MA) Sprint PowerScript reagents according to manufacturer’s
protocol. PCR amplification and analysis of PCR reaction were
Figure 2. 1D11 improved gross appearance of fibrotic livers. Representative photomicrographs at week 16 are shown. Gross examination of
livers taken from normal controls (A), PBS untreated group (B), and control antibody group (C) showed stiff, swelling livers with rough, granular or
nodular surface changes (yellowish in color) reminiscent of severe liver fibrosis in humans. Liver from rats that received 1D11 had much improved
gross appearance (D), showing smoother surface and less nodular changes.
TGF-b Neutralization in Hepatic Fibrosis
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Figure 3. 1D11 reversed pre-existing hepatic fibrosis. A. Representative photomicrographs of picrosirius red stained liver sections following
treatment with TAA for 8 weeks (‘‘baseline’’; TAA-8W), or an additional 8 weeks with 13C4 (13C4–16W) or 1D11 (1D11–16W) after cessation of TAA
administration. A normal rat liver is also shown (Normal). Livers of rats dosed with TAA for 8 weeks (TAA-8W) had substantial lesions with widely
spread fibrous bands (septa), originating from portal areas and extending into the parenchyma. 8 weeks after stopping TAA dosing, fibrotic areas
were further expanded, with extensive architectural disorganization and more fibrosis covering a greater percentage of the livers (13C4–16W).
Treatment with 1D11 for 8 weeks significantly ameliorated the TAA-mediated histopathological lesions, as shown by an overall improvement in
hepatic morphology (1D11–16W). Liver collagen deposition in rats treated with 1D11 was noticeably much reduced than the pre-established fibrosis
seen at baseline (TAA-W8), shown in rats that were examined at baseline. Magnification: 40x. B. Morphometric analysis of picrosirius red stained
sections. Significantly less collagen was deposited in livers treated with 1D11 (m) for 4 weeks or 8 weeks, when compared to the PBS (N, * p,0.01) or
13C4 (&, * p,0.01) treatment groups. Moreover, comparison of 1D11 treated livers to those at week 8 before therapeutic treatment started, showed
significantly less collagen deposition (treatment for 4 weeks: 9.61%; 8 weeks: 7.53%, * p,0.05) than that harvested at baseline (week 8:12.7%).
Morphometric quantification further supports the histology of a reversal of pre-existing hepatic fibrosis when TGF-b was neutralized by 1D11. Values
represent the percentage of collagen deposition in the total liver section and are expressed as mean 6 SE (n=8, except n=4 for normal controls at
TGF-b Neutralization in Hepatic Fibrosis
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performed and monitored using an ABI Prism 7700 Sequence
Detection System (TaqMan, Perkin-Elmer Applied Biosystem,
Carlsbad, CA). For each cDNA sample the Ct value of each target
sequence was normalized to ribosomal RNA-18S to obtain the
relative levels of mRNA expression of TGF-b. PCR reactions were
performed in a 96-well plate in the TaqMan system. Values are
expressed as fold change vs. normal controls.
Liver tissues were homogenized in ice-cold Tris lysis buffer (BD
Biosciences) containing 1% triton-X100 plus protease inhibitor
cocktail tablet (Roche Diagnostics, Indianapolis, IN); protein
concentration was measured by the BCA protein assay kit (Pierce,
Rockford, IL). Samples were boiled for 10 min and equal amounts
of protein were loaded onto NuPage 4–12% Bis-Tris gradient gel
(Invitrogen, Carlsbad, CA). Proteins were transferred onto a
nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ).
Blots were incubated in blocking buffer (1x TBS-0.1% Tween-20
with 5% (wt/vol) nonfat dry milk for 1 hour followed by
incubation of the blots with a mouse anti-PAI-1 antibody
(1:1000, BD Sciences) at 4uC over night. Blots were then washed
three times with washing buffer (1x TBS-0.1% Tween-20) and
incubated with a secondary HRP-conjugated donkey anti-mouse
IgG antibody (1:5000, Amersham Biosciences) for 1 hour at room
temperature. Bound antibody was visualized with the enhanced
chemiluminescence reagent kit (Amersham Bioscience). Protein
molecular weight standards (Invitrogen) were used to assess
Measurement of Total and Active PAI-1 Protein
Liver slices were homogenized at 4uC for 15 seconds at full
speed on an Ultra-Turrax tissue homogenizer. Protein concentra-
tion of the homogenates was determined by BCA protein assay
(Pierce). Total tissue type PAI-1 protein was determined using a
solid phase enzyme immunoassay with two complementary
murine monoclonal antibodies specific for PAI-1 (Hyphen
BioMed, Neuville-sur-Oise, France). Briefly, tissue homogenates
were incubated and bound to the first specific murine monoclonal
pre-coated on solid phase through one epitope, recognized with
the second specific antibody coupled to HRP. For active PAI-1
protein, tissue homogenates were incubated and bound to active
recombinant tPA pre-coated in an ELISA plate. Only active PAI-1
reacts with tPA and is fixed on the solid phase. After washing, a
mouse monoclonal antibody specific for human PAI-1 and
coupled to HRP was applied and bound to active PAI-1. PAI-1
protein was measured at 450 nm, with a recombinant human PAI-
1 used as a standard. Values were expressed as pg/mg protein.
Immunohistochemistry for cytokeratin (CK), a biliary epithelial
marker , was carried out on deparaffinized liver sections.
Briefly, sections were first treated at 85uC with an unmasking
solution containing 1% of sodium citrate (Vector Laboratories,
Burlingame, CA) and then incubated with a universal protein
blocking reagent (DAKO, Carpinteria, CA) for 30 minutes at
room temperature. Sections were then incubated for 1 hour with a
rabbit polyclonal antibody against pan-CK (1:100 dilution,
DAKO). In our testing, this antibody was verified to recognize
only biliary epithelial cells, without non-specific staining to other
cell types or interstitial components. After washing, sections were
incubated for 45 minutes with horseradish peroxidase (HRP)
conjugated polymer anti-rabbit secondary antibody followed by
incubation with DAB chromogen as a substrate (DAKO) for 1
minute. Sections were counter-stained with hematoxylin to
visualize hepatic architecture.
Values are presented as mean 6 SE. Statistical analysis of values
was performed using One-Way ANOVA or the unpaired student t
test, with P values ,0.05 considered significant.
Clinical Findings and Blood Chemistry Analysis
TAA dosing resulted in 13% mortality, which occurred
primarily in the first 2 to 3 weeks of TAA administration. TAA
dosing also caused a loss of body weight, which upon cessation of
administration, was restored within 6 weeks to levels equivalent to
normal controls regardless of PBS, 13C4 or 1D11 treatment (data
not shown). Furthermore, during the time of TAA administration,
there was corresponding evidence from the hepatic injury markers,
ALT and AST, of cytotoxic insult, in agreement with published
reports . Upon cessation of TAA dosing, plasma liver enzymes
Table 1. 1D11 significantly reduces hepatic hydroxyproline
Hydroxyproline content (ug/mg dry weight)
Normal controls0. 5060.02
Animals were dosed with PBS, 13C4, and 1D11 for 8 weeks after cessation of
*p,0.05 vs. normal controls and ** p,0.05 vs. PBS, 13C4 and normal control
groups. Values represent mean 6 SE.
Figure 4. 1D11decreased TGF-b1 mRNA. mRNA expression of TGF-
b1 was analyzed by quantitative real-time RT-PCR for livers harvested at
baseline (prior to treatment) and at week 16 (after treatment). TAA
caused a sustained overexpression of TGF-b1 (.6 fold) throughout the
study and treatment with 1D11 significantly reduced mRNA levels
compared to baseline levels and in both PBS and 13C4 groups at end of
study (week 16). Data at week 16 with 1D11 treatment suggest a
reversal of fibrosis. Values are expressed as means 6 SE, n=8 per
group; * p,0.01 vs. normal controls at week 8; ** p,0.05 vs. PBS or
13C4 group at week 16.
TGF-b Neutralization in Hepatic Fibrosis
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over the course of 6 weeks returned to within normal levels (data
not shown) and there were no significant differences among the
PBS, 13C4 or 1D11 groups during this time (data not shown).
Administration of 1D11 and 13C4 had no observable deleterious
effect on the general health of animals during the period of
investigation. No death of animals was found due solely to the
administration of antibodies.
Gross Appearance, Histopathological Assessment and
Gross examination showed stiff, swelling livers with rough,
granular or nodular changes (yellowish in color) on the surface
(Figure 2), resembling severe liver fibrosis in humans. Rats treated
with 1D11 had much improved gross appearance (Figure 2D),
showing smoother surface and less nodular change, as compared
to rats dosed with PBS or 13C4 (Figure 2B and C). Histologic
analysis showed widely spread fibrous bands (septa), originating
from portal areas and extending into the hepatic parenchyma of
rats upon cessation of TAA dosing (Figure 3A, TAA-8W). The
disease groups, dosed with PBS (data not shown) or 13C4, showed
further deterioration of hepatic architecture; portal fibrotic foci
were more pronounced, with more apparent bridging fibrosis
covering a greater percentage of the hepatic parenchyma.
Treatment with 1D11 for 4 (data not shown) or 8 weeks
significantly reduced TAA-induced collagen deposition/bridging
fibrosis, and other lesions, as shown by an overall improvement in
hepatic morphology (Figure 3A; 1D11-16w). The histologic
appearance of samples from the 1D11 treated group also exhibited
an improvement over baseline histology at the time point when
Figure 5. Increased PAI-1 protein in fibrotic liver was reduced in rats dosed with 1D11. A. A representative Western blot of pooled
samples (n=3) showed marked increase in PAI-1 expression before the start of therapeutic dosing at week 8. Levels were maintained in both PBS and
13C4 groups at the end of study at week 16. Levels of PAI-1 were normalized in livers treated with 1D11. B and C. Hepatic PAI-1 protein was also
analyzed by ELISA. At the end of TAA administration, total (B) and active PAI-1 (C) were markedly elevated in fibrotic livers, with a further increase by
week 16 in rats treated with PBS or 13C4. After 8 weeks of 1D11 dosing, total PAI-1 and active PAI-1 had returned to normal levels, confirming the
immunoblotting data. Values are expressed as mean 6 SE, n=8; * p,0.01 vs. normal controls at week 8; ** p,0.01 vs. PBS or 13C4 group at week 16.
TGF-b Neutralization in Hepatic Fibrosis
PLOS ONE | www.plosone.org6January 2013 | Volume 8 | Issue 1 | e54499
TAA was stopped (Figure 3A; TAA-8w). These data strongly
suggest that 1D11 was efficacious in treating pre-existing hepatic
fibrosis and suggest some reversibility of histologically apparent
fibrosis. Conventional blinded semi-quantitative scoring showed
that rats dosed for 8 weeks with 1D11 had lower pathological
scores (1.960.05), compared with those treated with PBS
(3.460.1) or 13C4 (3.660.2). Morphometric analysis of liver
sections (Figure 3B) revealed quantitative reduction in collagen
deposition area (expressed as percentage of liver sections) in rats
dosed with 1D11 for 4 (9.61%, p,0.05) or 8 weeks (7.53%,
p,0.05) as compared to pre-existing fibrosis established before
antibody treatment (12.65%). Progressive collagen deposition
occurred in the control PBS and 13C4 groups. For comparison,
the percent picrosirius red stain in normal liver was 2.1460.5 at
week 0 and 2.5260.26 at week 16. The analysis of picrosirius red
staining coupled with blinded histopathologic scoring suggests that
Figure 6. 1D11 reduced cholangiocarcinomas induced by TAA. A. TAA induced profound neoplastic changes in biliary ductules
(cholangiocarcinoma) at week 16. As visualized by CK staining, these neoplastic cells extended into the parenchyma, as fibrosis evolved (arrow
heads). The neoplasia displayed a typical ‘‘intestinal metaplasia’’-like appearance (arrows). The number of neoplastic biliary ductules or cells was much
lower with 1D11 treatment, compared to the PBS or 13C4 groups. For comparison, a normal liver stained for CK is shown. Magnification: x100 B. CK
staining was quantitated. The TAA-induced neoplasia was significantly reduced in rats treated with 1D11 for 8 weeks. In contrast, the neoplasia
showed no reduction in rats dosed with PBS or 13C4. Values are expressed as mean 6 SE of percentage of CK staining of the entire area of liver
section. n=8; * p,0.01 vs. normal controls; ** p,0.01 vs. PBS, 13C4 and normal control groups. C. To quantify the areas of cholangiocarcinoma, liver
sections from the 16 time point were scanned by ChromaVision Imaging Analysis System. The area of the cholangiocarcinomas defined as neoplastic
biliary epithelial cells plus stromal fibrotic tissue and infiltrated cells, was measured and expressed as a percentage of the total area of the liver
section. Data revealed a striking reduction with 1D11 treatment, as compared to the PBS and 13C4 groups, reflecting a diminishment of
cholangiocarcinoma by TGF-b neutralization. n=8, * p,0.01 vs. normal controls; ** p,0.01 vs. PBS, 13C4 and normal control groups. Values
represent mean 6 SE.
TGF-b Neutralization in Hepatic Fibrosis
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therapeutic neutralization of TGF-b can block progressive hepatic
fibrosis and may also reverse existing fibrosis. Hepatic hydroxy-
proline content was also measured in this study. It was significantly
higher in rats treated with TAA followed with either PBS or 13C4
in comparison to normal controls (Table 1). TAA rats dosed with
1D11 had a significantly lower hydroxyproline content (1.0460.09
ug/dry tissue) than either the 13C4 or PBS group (p,0.05). These
data are consistent with and further support histological and
Effect of 1D11 on TGF-b1 Overexpression
Hepatic expression of TGF-b1 was analyzed by real-time RT-
PCR. At baseline (week 8) TGF-b1 expression was elevated 6 fold
(Figure 4) with similar increases in the other two isoforms (data for
TGF-b2 and b3 not shown). Interestingly, b3 rapidly returned to a
level of expression slightly above normal upon cessation of TAA
dosing, whereas TGF-b1 and b2 remained upregulated through-
out the end of the study. TGF-b1 expression was significantly
reduced with 1D11 dosing for 4 weeks (data not shown) or 8 weeks
(p,0.05). Expression in control treatment groups were slightly, but
not significantly reduced. These data suggest possible interruption
of the known TGF-b autocrine regulation loop . In contrast,
TGF-b2 remained unchanged upon TGF-b neutralization (data
not shown), suggesting that this isoform might not be accessible by
1D11, due to an intracellular localization in biliary epithelial cells
(data not shown).
Effect of 1D11 on Elevation of PAI-1
Elevation of PAI-1 may play a key role not only in fibrinolytic
but also extracellular matrix turnover [9–10]. Because neutraliza-
tion of TGF-b appeared to reverse existing hepatic fibrosis, we
examined PAI-1 expression in this model. The expression of PAI-1
protein in livers was analyzed by Western blot, in which individual
or pooled samples of liver homogenates from all treatment groups
were assessed. A representative Western blot of pooled samples
(n=3) shows that PAI-1 protein was significantly increased at
baseline and remained elevated in both control groups through the
end of study (Figure 5A). In contrast, PAI-1 levels were nearly
undetectable in rats dosed with 1D11. These findings are
consistent with published data suggesting that PAI-1 over-
expression is associated with suppressed ECM degradation in
organ fibrosis [10–11,25]. Further supporting this notion was the
quantitative measurement of total PAI-1 (Figure 5B) and also
active PAI-1 (Figure 5C) as determined in liver homogenates by
ELISA. Interestingly, the increase in both total and active PAI-1
levels coincided with the progression of fibrosis, and was further
elevated in both control groups at end of study. 1D11 dosing
normalized both total PAI-1 and active PAI-1.
Effects of 1D11 on TAA-induced CCA
TAA is also a potent carcinogen which induces CCA over a
course of 10 to 22 weeks in rats [20–21]. In the present study,
extensive microfoci of neoplastic biliary ductules embedded in
fibrotic tissues were induced around week 8–12, and mass-forming
CCA were seen at 16 weeks, which extended, as fibrosis evolved,
into the parenchyma. The biliary neoplasia displayed a typical
‘‘intestinal metaplasia’’-like appearance, with mucins found inside
biliary epithelial cells or secreted into the lumen (Figure 6A). To
better visualize and optimally quantify the area of these neoplastic
biliary ductules, a monoclonal antibody against cytokeratin, an
epithelial-specific marker, was applied using immunohistochemis-
try staining and qualified by morphometric analysis. All neoplastic
biliary ductules, were immunostained, with no evidence of non-
specific staining to other cell types or interstitial components. The
number of ductules (or cells) was reduced substantially (45%) in
rats dosed with 1D11 compared to the PBS or 13C4 control
groups (Figure 6B) at study end. An average of 15 to 25 individual
neoplastic foci were detected in rats from PBS or 13C4 control
groups, whereas 3–7 foci were detected in 1D11 treated rats. To
further quantify the area of CAA consisting largely of neoplastic
biliary ductules and fibrotic stromal tissues, individual liver
sections were qualified by morphometric analysis. This analysis
(Figure 6C) showed a significant reduction in the area of CCA in
rats treated with 1D11, as compared to the PBS or 13C4 treated
control groups (p,0.01).
Numerous experimental and clinical studies have demonstrated
a central role of TGF-b, and in particular b1, in liver fibrosis [7–
9]. Studies using engineered TGF-b soluble receptors or siRNA
interference technique have directly confirmed a causative role of
this cytokine in liver fibrosis [16–19]. While the evidence linking
TGF-b and fibrogenesis is substantial and raises possibilities for
targeting this molecule as an appropriate therapeutic agent, it is
necessary to demonstrate whether TGF-b antagonism can
effectively treat pre-existing hepatic fibrosis. In the present study,
we treated healthy rats with TAA for 8 weeks to establish hepatic
fibrosis, which was histologically close to that seen in human liver
cirrhosis. With fibrosis established, we examined the effect of
administering a TGF-b neutralizing antibody, 1D11, on further
fibrosis progression, and also whether there was evidence of
regression from pre-existing fibrosis. After TAA withdrawal both
control groups showed further deterioration of liver architecture
mostly in the form of nodular damage and fibrous foci or septa
bridging from portal area to portal area and to central areas. Also
noted was substantial evolution of proliferating biliary epithelial
neoplasia (CAA) within fibrous foci, consistent with earlier reports
of TAA mediated hepatic lesions [20–21]. The degree of TAA-
induced lesions correlated with the expression of TGF-b mRNA in
the liver. TGF-b1, b2 and b3 were significantly upregulated after 8
weeks of TAA administration (6-fold increase for b1), and
remained high in rats that received PBS or 13C4, except for
TGF-b3, which declined but was still higher than normal controls
upon the cessation of TAA administration.
Treatment with 1D11 reduced transcription of TGF-b1whereas
the other two isoforms were unchanged, most likely due to their
intracellular location in biliary epithelia. Reduced TGF-b1
expression and apparent interruption of the TGF-b1 autocine
regulatory loop likely explain the therapeutic impact on pre-
existing hepatic fibrosis. Histologically, a profound improvement
in hepatic architecture with reduced number and area of fibrous
foci or septa was seen in rats dosed with 1D11 for 8 weeks.
Morphometric analysis, along with the measurement of hydroxy-
proline content, showed a significant decrease in collagen
deposition in animals treated with 1D11. Of note, the two control
groups showed progressive hepatic injury 4 and 8 weeks following
cessation of TAA-induced injury, whereas the 1D11 treated group
showed regression of fibrosis, significantly lower than levels
recorded just before starting 1D11 treatment. Thus in this model
there was no evidence of spontaneous resolution of injury, unlike
other models  and, further, our data argue that antibody-
mediated antagonism of TGF-b promotes regression of tissue
fibrosis. We conclude that pathological hepatic fibrosis, at least in
this preclinical model, is a dynamic situation and is potentially
reversible. This may also be true in the human since recent studies
examining hepatitis patients with long term antiviral therapy
showed reduced liver cirrhosis [27–28]. Our results provided a
TGF-b Neutralization in Hepatic Fibrosis
PLOS ONE | www.plosone.org8 January 2013 | Volume 8 | Issue 1 | e54499
platform for further mechanistic studies focusing on the revers-
ibility of pathological fibrosis.
Accumulation of ECM proteins is caused not only by increased
protein synthesis but also by decreased protein degradation. There
is now a significant body of evidence that liver fibrosis is also a
consequence of altered ECM degradation, in which PAI-1 may be
implicated [10–11]. PAI-1 is the major physiologic inhibitor of
tissue-type (t-PA) or urokinase-type plasminogen activator (m-PA),
both of which activate plasminogen to plasmin. Plasmin may also
activate unrelated proteases to further promote resolution of
fibrosis . In the present study, a marked increase of PAI-1
protein occurred in parallel with fibrosis progression. PAI-1
remained elevated in rats treated with PBS or 13C4, but this was
normalized in rats dosed with a TGF-b neutralizing antibody.
Thus our data suggest that elevated PAI-1 contributed to the
development of hepatic fibrosis through a TGF-b dependent
manner. These data are consistent with several recent studies
showing that; 1) direct manipulation of plasmin/PAI-1 with
adenovirus-mediated transfer of siRNA decreased hepatic fibrosis
in dimethylnitrosamine-induced and bile duct ligation-induced
liver disease models ; 2) restoration of hepatic plasmin activity
by a mutant, noninhibitory PAI-1 was also associated with
decreased fibrotic matrix accumulation in this model  and,
3) higher levels of tPA activation have been associated with fewer
hepatic lesions in PAI-1 (2/2) mice . Reduced PAI-1 protein,
along with attenuated TGF-b1 transcription may indicate an
inhibition of TGF-b1/Smad signaling axis by 1D11, presumably
with decreased phosphorylation of Smad2/3, a principal effector
system of the signaling pathway contributing to fibrosis .
It is also known that TAA is a potent carcinogen and induces
CCA in the rat, which recapitulates the histological features and
progression of human CCA [20–21]. In the present study, TAA
induced hepatic fibrosis, concomitant with development of
neoplastic biliary epithelial ductules embedded in fibrotic/stromal
tissues. The nature of this type of neoplastic biliary ductules was
confirmed morphologically as a typical ‘‘intestinal metaplasia’’-like
appearance, identified by immunostaining of cytokeratins ,
and microscopic diagnosis by a board certified pathologist. The
assessment of TAA-induced CCA by immunostaining also
provided a way to determine the area of the neoplastic biliary
ductules. Neoplastic bile ductules were remarkably fewer in
number and in area in livers from rats dosed with 1D11. The
diminishment of CCA occurred in parallel with an improved
fibrosis index, suggesting a close relationship between these two.
Indeed, elaboration of ECM proteins provides both a three-
dimensional structure as well as matrix-cellular signals that
promote tumorigenesis [16,33–34]. With specific regard to
CCA, Farazi PA et al.  have shown that increased production
of type I and III collagens along with fibroblast recruitment
stimulate biliary epithelium hyperplasia and subsequent progres-
sion to malignant intrahepatic tumors mice harboring a p53
mutant allele. Thus our findings are consistent with others’
observations suggesting that therapeutic reduction of hepatic
fibrosis as a result of TGF-b neutralization represents a potential
approach for the treatment of CCA.
To summarize, the present study demonstrates that 1D11, a
murine monoclonal TGF-b neutralizing antibody, can reverse pre-
existing clinically comparable hepatic fibrosis induced by an
extended dosing of TAA. The regression of fibrosis was
accompanied with a diminishment of cholangiocarcinoma, pre-
sumably linked to a reduction of fibrosis surrounding neoplastic
ductules, associated with an inhibition of TGF-b and PAI-1. The
data provide the first evidence that reversal of pre-existing hepatic
fibrosis can be achieved upon TGF-b neutralization, providing
proof-of-concept for considering TGF-b neutralizing antibody in
the treatment of liver cirrhosis.
We thank Matthew McCourt and Nicola Davis at Cambridge Antibody
Technology (Cambridge, UK) for their excellent technical assistance in
PAI-1 assay. We are also grateful to Dr. Beth Thurberg (Genzyme) for
reviewing the histopathology of TAA-induced neoplastic biliary epithelial
cells and Brian DelGiudice (Genzyme) for his assistance in figure
Conceived and designed the experiments: HL S. Lonning S. Ledbetter.
Performed the experiments: HL ER DH JT. Analyzed the data: HL ER
DH JT MS. Contributed reagents/materials/analysis tools: HL ER DH JT
MS. Wrote the paper: HL AZ CA S. Ledbetter.
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