Reduced Liver Fibrosis in Hypoxia-inducible Factor-1α α-Deficient Mice
Jeon-OK Moon1,2, Timothy P. Welch1, Frank J. Gonzalez3 and Bryan L. Copple1
1Department of Pharmacology, Toxicology, and Experimental Therapeutics
University of Kansas Medical Center, Kansas City, KS 66160
2Department of Pharmacy, Pusan National University, Busan 609-735, South Korea
3Laboratory of Metabolism, Center for Cancer Research, National Cancer
Institute, National Institutes of Health, Bethesda, MD
Running Head: Role of HIF-1α in Liver Fibrosis
Contact Information for Corresponding Author:
Bryan L. Copple, Ph.D.
Department of Pharmacology, Toxicology, and Therapeutics
University of Kansas Medical Center
3901 Rainbow Boulevard
Kansas City, KS 66160
Articles in PresS. Am J Physiol Gastrointest Liver Physiol (January 8, 2009). doi:10.1152/ajpgi.90368.2008
Copyright © 2009 by the American Physiological Society.
Liver fibrosis is characterized by excessive deposition of extracellular matrix in
the liver during chronic injury. During early stages of this disease, cells begin to
synthesize and secrete profibrotic proteins that stimulate matrix production and inhibit
matrix degradation. Although it is clear that these proteins are important for
development of fibrosis, what remains unknown is the mechanism by which chronic liver
injury stimulates their production. In the present study, the hypothesis was tested that
hypoxia-inducible factor-1α (HIF-1α) is activated in the liver during chronic injury and
regulates expression of profibrotic proteins. To investigate this hypothesis, mice were
subjected to bile duct ligation (BDL), an animal model of liver fibrosis. HIF-1α protein
was increased in the livers of mice subjected to BDL by 3 days after surgery. To test
the hypothesis that HIF-1α is required for the development of fibrosis, Control and HIF-
1α-deficient mice were subjected to BDL. Levels of type I collagen and α-smooth
muscle actin mRNA and protein were increased in Control mice by 14 days after BDL.
These levels were significantly reduced in HIF-1α-deficient mice. Next, the levels of
several profibrotic mediators were measured to elucidate the mechanism by which HIF-
1α promotes liver fibrosis. Platelet-derived growth factor (PDGF)-A, PDGF-B, and
plasminogen activator inhibitor-1 mRNA levels were increased to a greater extent in
Control mice subjected to BDL when compared to HIF-1α-deficient mice at 7 and 14
days after BDL. Results from these studies suggest that HIF-1α is a critical regulator of
profibrotic mediator production during the development of liver fibrosis.
Bile duct ligation
Platelet-derived growth factor
α-smooth muscle actin
Liver fibrosis is characterized by excessive deposition of extracellular matrix,
such as collagen, in the liver during chronic injury. This disease is initiated when liver
injury stimulates cells to synthesize and secrete growth factors, such as platelet-derived
growth factor and other soluble mediators that activate hepatic stellate cells and
stimulate them and other cell types, such as peribiliary fibroblasts to produce collagen
(10, 21). In addition, other mediators are produced, such as matrix metalloproteinases
that modulate matrix turnover (14, 22). Although it is clear that numerous mediators are
important for the initiation and propagation of fibrosis, what remains largely unknown is
the mechanism by which liver injury stimulates their production. A greater
understanding of the mechanism(s) that regulate profibrotic mediator production, may
lead to the development of novel treatments for this disease. One factor that may
promote production of profibrotic mediators is hepatocellular hypoxia.
Several studies have demonstrated that the liver becomes hypoxic when injured
(8, 9, 18, 28). Hypoxia likely results from disrupted hepatic blood flow and sinusoidal
fibrin deposition (8). Furthermore, changes occur in patients with liver disease that may
promote hepatocellular hypoxia, such as systemic hypoxemia, sinusoidal capillarization,
and formation of portal-systemic collateral vessels and intrahepatic shunts. Hypoxia
has been shown to modulate gene expression by activating a number of transcription
factors, including hypoxia-inducible factors (HIFs). HIFs are composed of an alpha
subunit, typically HIF-1α or HIF-2α, and a beta subunit, HIF-1β also called the
arylhydrocarbon receptor nuclear translocater (7, 11). In normoxic cells, HIFα subunits
are constitutively produced and immediately targeted for proteolytic degradation. When
cells become hypoxic, however, the mechanisms that target HIFα subunits for
degradation are inhibited allowing HIFα protein levels to increase (5). HIFα subunits
then translocate to the nucleus where they heterodimerize with HIF-1β and regulate
expression of genes involved in glycolysis, angiogenesis, iron metabolism, pH control,
and others functions. Interestingly, many of the genes that HIFs regulate, including
various growth factors, have been implicated in the pathogenesis of liver fibrosis.
Whether HIFs are activated in liver during fibrosis and regulate expression of genes that
promote this disease, however, has not been examined.
The overall goal of the studies presented in this manuscript was to determine the
mechanism by which profibrotic mediators, such as platelet-derived growth factor
(PDGF), fibroblasts growth factor-2 (FGF-2), and plasminogen activator inhibitor-1 (PAI-
1) are upregulated in the liver during chronic injury. The hypothesis was tested that
during chronic liver injury, HIF-1α is activated and promotes liver fibrosis by regulating
expression of genes, such as PDGF, FGF-2, and PAI-1 that are critical for the genesis
Materials and Methods
Mice. Deletion of HIF-1α in mice causes embryonic lethality (17). To selectively
reduce HIF-1α levels in adult mice, HIF-1αfl/fl mice, described in detail previously (31)
were crossed with mice expressing Cre recombinase under control of the Mx interferon-
inducible promoter (23) (Mx-Cre+/- mice; Jackson Laboratories, Bar Harbor, ME).
Offspring of this breeding were HIF-1αfl/fl-Mx-Cre+ (i.e., HIF-1α-deletable) or HIF-1αfl/fl-
Mx-Cre- (i.e., HIF-1α-nondeletable littermate controls). PCR of genomic DNA was used
to detect the floxed HIF-1α gene and the Cre transgene as described previously (31).
To activate the MxCre promoter, HIF-1αfl/fl-Mx-Cre+ and HIF-1αfl/fl-Mx-Cre- mice were
treated with 500 µg of polyinosinic–polycytidylic acid (pIpC, Sigma Chemical Company,
St. Louis, MO) dissolved in sterile saline by intraperitoneal injection every three days for
a total of three injections. In mice containing the MxCre transgene, this treatment
causes a near complete deletion of loxP-containing genes in liver and immune organs
and a partial deletion in other tissues (23, 30). In HIF-1αfl/fl-Mx-Cre+ mice, this
treatment results in the deletion of exons 13-15 from HIF-1α which contain the COOH-
terminal transactivation domain and a portion of the nuclear localization sequence, both
of which are essential for hypoxia responsiveness by HIF-1α (31). Mice were used for
experiments one week after the final pIpC injection.
All mice were maintained on a 12-h light/dark cycle under controlled temperature
(18-21ºC) and humidity. Food (Rodent Chow; Harlan-Teklad, Madison, WI) and tap
water were allowed ad libitum. All procedures on animals were carried out in
accordance with the Guide for the Care and Use of Laboratory Animals promulgated by
the National Institutes of Health.
Analysis of HIF-1α α Deletion in Liver. Genomic DNA was isolated from the
livers of mice and PCR was used to determine the degree of HIF-1α deletion. PCR was
performed using the following primers: Primer HI: 5'-CTG TCT TCC CTG CTT AGG
TCT TTC TAA C-3', Primer H2: 5'-GAG ATG GAG AAG GAG GTT AGT GTA TCC-3',
and Primer H3: 5'-ACG TTG GCT CAT GGT GTA CTT TG-3' as described previously
Bile Duct Ligation. Male, C57BL/6 (Harlan, Indianapolis, IN), HIF-1αfl/fl-Mx-
Cre+, and HIF-1αfl/fl-Mx-Cre- mice 8-12 weeks of age were anesthetized with isoflurane.
A midline laparotomy was performed and the bile duct ligated with 3-0 surgical silk. The
abdominal incision was closed with sutures, and the mice received 0.2 mg/kg Buprenex
by subcutaneous injection.
Real-time PCR. Total liver RNA was isolated using TRI reagent (6) (Sigma
Chemical Company, St. Louis, MO), and reverse transcribed into cDNA as described by
us previously (20). Real-time PCR was used to quantify the mRNA levels of collagen
type Iα1, α-smooth muscle actin (α-SMA), 18S, platelet-derived growth factor (PDGF)-
A, PDGF-B, connective tissue growth factor (CTGF), fibroblast growth factor-2 (FGF-2),
and plasminogen activator inhibitor-1 (PAI-1) on an Applied Biosystems Prism 7300
Real-time PCR Instrument (Applied Biosystems, Foster City, CA) using the SYBR green
DNA PCR kit (Applied Biosystems) as described (20). The sequences of the primers
were as follows: 18S Forward: 5’-TTG ACG GAA GGG CAC CAC CAG-3’; 18S
Reverse: 5’-GCA CCA CCA CCC ACG GAA TCG-3’; α-SMA Forward: 5’-CCA CCG
CAA ATG CTT CTA AGT-3’; α-SMA Reverse: 5’-GGC AGG AAT GAT TTG GAA AGG-
3’; collagen type Iα1 Forward: 5’-TGT GTT CCC TAC TCA GCC GTC T-3’; collagen
type Iα1 Reverse: 5’-CAT CGG TCA TGC TCT CTC CAA-3’; CTGF Forward: 5’-CTG
CCA GTG GAG TTC AAA TGC-3’; CTGF Reverse: 5’-TCA TTG TCC CCA GGA CAG
TTG-3’; FGF-2 Forward: 5’-AGC GAC CCA CAC GTC AAA CTA C-3’; FGF-2 Reverse:
5’-CAG CCG TCC ATC TTC CTT CAT A-3’; PAI-1 Forward: 5’-AGT CTT TCC GAC
CAA GAG CA-3’; PAI-1 Reverse: 5’-ATC ACT TGC CCC ATG AAG AG-3’; PDGF-A
Forward: 5’-GAG ATA CCC CGG GAG TTG AT-3’; PDGF-A Reverse: 5’-AAA TGA
CCG TCC TGG TCT TG-3’; PDGF-B Forward: 5’-CCC ACA GTG GCT TTT CAT TT-3’;
PDGF-B Reverse: 5’-GTG GAG GAG CAG ACT GAA GG-3’.
Western Blot Analysis. A portion of liver was homogenized in 10 mM Hepes,
pH 7.9 containing 100 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM DTT, 0.5% NP-
40, and Halt Protease Inhibitor Cocktail (Pierce Biotechnology, Rockford, IL) and
incubated on ice. After 15 minutes, the homogenate was vortexed and centrifuged for
10 seconds at 10,000 g. The pellet was resuspended in 10 mM Hepes, pH 7.9
containing 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM DTT, 5% glycerol, and
Halt Protease Inhibitor Cocktail mixed for 1 hour at 4°C. The samples were centrifuged
at 10,000 g for 10 minutes at 4°C and the concentration of protein determined in the
supernatant. Aliquots (15 µg) of nuclear extracts were subjected to 10% SDS-
polyacrylamide gel electrophoresis, and proteins were transferred to Immobilon
polyvinylidene difluoride transfer membranes (Millipore Corporation, Bedford, MA). The
membranes were then probed with rabbit polyclonal anti-HIF-1α antibody (NB100-449,
Novus Biologicals, Littleton, CO) diluted 1:1000, followed by incubation with goat anti-
rabbit antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology).
Immunoreactive bands were visualized using the Immun-Star HRP Substrate Kit (Bio-
Rad Laboratories, Hercules CA).
HIF-1α α Immunohistochemistry. For HIF-1α immunostaining, livers were frozen
in isopentane (Sigma Chemical Company) immersed in liquid nitrogen for 8 minutes.
Sections of frozen liver were fixed in 4% formalin in phosphate-buffered saline (PBS) for
10 min at room temperature. Sections were incubated with rabbit polyclonal anti-HIF-1α
antibody (NB100-449, Novus Biologicals, Littleton, CO) diluted 1:50 in PBS containing
3% goat serum at room temperature for 3 h. The sections were washed with PBS, and
then incubated with secondary antibody conjugated to Alexa 488 (green staining;
Invitrogen). The sections were counterstained with Alexa Fluor 594-conjugated
phalloidin to stain actin. For double immunohistochemistry for HIF-1α and
macrophages, sections of liver were incubated with anti-HIF-1α antibody as described
above and rat anti-mouse F4/80 and rat anti-mouse CD68. The sections were washed
with PBS, and then incubated with anti-rabbit secondary antibody conjugated to Alexa
594 (red staining; Invitrogen) and anti-rat secondary antibody conjugated to Alexa 488
(green staining; Invitrogen). For double immunohistochemistry for HIF-1α and
hepatocytes, sections of liver were incubated with anti-HIF-1α antibody as described
above and goat anti-mouse albumin. The sections were washed with PBS, and then
incubated with anti-rabbit secondary antibody conjugated to Alexa 594 (red staining;
Invitrogen) and anti-goat secondary antibody conjugated to Alexa 488 (green staining;
Pimonidazole Immunohistochemistry. To detect regions of hypoxia in the
liver, mice were treated with pimonidazole (120 mg/kg, Chemicon, Temecula, CA)
dissolved in sterile saline by intraperitoneal injection 1, 3, 7, or 14 days after BDL and
were killed 1 hour later. Immunostaining for pimonidazole was performed as described
Hypoxia in the liver was quantified morphometrically by analyzing the area of
immunohistochemical staining of pimonidazole in a section of liver using Scion Image
software (Scion Corporation, Frederick, MD) as described (8). An increase in the area
of pimonidazole staining in the liver indicates hypoxia. The staining is expressed as a
fraction of the total area. The random fields analyzed for each liver section were
averaged and counted as a replicate, i.e., each replicate represents a different mouse.
For the time-course studies, data from mice subjected to sham operation at various
times were combined into one group for statistical analysis, since no differences
occurred among sham-operated groups.
Cytokeratin 19 Immunohistochemistry. For cytokeratin 19 (CK19)
immunostaining, livers were frozen in isopentane (Sigma Chemical Company)
immersed in liquid nitrogen for 8 minutes. Sections of frozen liver were fixed in 4%
formalin in phosphate-buffered saline (PBS) for 10 min at room temperature. Sections
were incubated with rat anti-CK19 antibody (Devlopmental Studies Hybridoma Bank,
Iowa City, IA) diluted 1:1000 in PBS containing 3% goat serum at room temperature for
3 h. The sections were washed with PBS, and then incubated with secondary antibody
conjugated to Alexa 488 (green staining; Invitrogen). The area of CK19 immunostaining
was quantified morphometrically as described previously (8).
Assessment of Biomarkers of Hepatic Injury and Cholestasis. Hepatocyte
injury and cholestasis were evaluated by measuring the activity of alanine
aminotransferase (ALT) and alkaline phosphatase (ALP) in the serum (Pointe Scientific
Inc., Brussels, Belgium). Serum bile acid concentrations were determined by using a
commercially available kit (Colorimetric Total Bile Acids Assay Kit; Bioquant, San Diego,
Quantification of Type I Collagen in the Liver. Type I collagen in the liver
was detected using immunohistochemistry and quantified morphometrically by
analyzing the area of immunohistochemical staining of type I collagen as described by
us previously (20). An increase in the area of type I collagen staining in the liver is an
indicator of fibrosis. The staining is expressed as a fraction of the total area. The
random fields analyzed for each liver section were averaged and counted as a replicate,
i.e., each replicate represents a different mouse.
Statistical Analysis. Results are presented as the mean + SEM. Data were
analyzed by Analysis of Variance (ANOVA). Data expressed as a fraction were
transformed by arc sine square root prior to analysis. Comparisons among group
means were made using the Student-Newman-Keuls test. The criterion for significance
was p < 0.05 for all studies.
Hypoxia in the Livers of Mice after Bile Duct Ligation. To determine whether
cells in the liver become hypoxic during the development of liver fibrosis, mice were
injected with pimonidazole. In cells that are hypoxic (i.e., <10 mmHg oxygen),
pimonidazole is metabolized to a reactive intermediate that binds to sulfur containing
constituents within cells (1). Immunohistochemistry can then be used to detect the
pimonidazole-sulfur adducts, which only occur in hypoxic cells. In liver sections from
mice subjected to sham operation, no immunohistochemical staining for hypoxia (i.e.,
positive pimonidazole staining) was observed at 3 days after surgery (Fig. 1A). Staining
for hypoxia was present in the livers of mice subjected to bile duct ligation 3 days earlier
(Fig. 1B). At this time, hypoxia was primarily present at the periphery of bile infarcts
(i.e., regions of hepatocellular necrosis extending from periportal areas). The extent of
hypoxia increased at later times. At no time-point, however, was hypoxia observed in
periportal regions where bile duct proliferation occurred (i.e., areas of ductular reaction).
Hypoxia was restricted to the hepatic parenchyma.
Morphometric analysis revealed that bile duct ligation caused a time-dependent
increase in the area of hypoxia staining in the liver that was significant by 3 days after
surgery (Fig. 1C).
Activation of HIF-1α α in the Liver after Bile Duct Ligation. Nuclear extracts
were isolated from the livers of mice subjected to sham operation or bile duct ligation,
and western blot analysis was performed to detect HIF-1α. Bile duct ligation increased
HIF-1α protein by 4-fold at 3 days after bile duct ligation and 7-fold by 7 days after bile
duct ligation (Fig. 2).
Immunohistochemistry revealed minimal HIF-1α in the livers of sham-operated
mice (Fig. 3A). Occasionally, positively stained hepatocytes were observed adjacent to
central veins. Numerous cells in the livers of mice subjected to bile duct ligation 7 days
earlier showed positive nuclear staining for HIF-1α (Fig 3B, C). These cells were both
adjacent to bile infarcts and present within periportal regions where bile duct
proliferation occurred. Green immunofluorescence occurring within the bile infarcts was
nonspecific and occurred in tissue sections incubated with only the fluorescent
Next, double immunohistochemistry was used to identify cell types in which HIF-
1α was activated. HIF-1α was activated in macrophages (Fig. 4A, B) and hepatocytes
(Fig. 4C, D) in sections of liver from bile duct ligated mice.
Effect of HIF-1α α Deletion on Markers of Cholestasis and Liver Injury. To
evaluate the role of HIF-1α in the development of liver fibrosis, we first determined the
deletion efficiency of HIF-1α in HIF-1αfl/fl-Mx-Cre+ mice and HIF-1αfl/fl-Mx-Cre- mice
treated with pIpC. One week after the final pIpC injection, DNA was isolated from liver
and the extent of HIF-1α deletion analyzed using PCR. As shown in Figure 5, HIF-1α
was deleted from 90-100% in HIF-1αfl/fl-Mx-Cre+ mice treated with pIpC. As expected,
no HIF-1α deletion was detected in HIF-1αfl/fl-Mx-Cre- mice treated with pIpC. For the
remainder of this manuscript, HIF-1αfl/fl-Mx-Cre+ mice treated with pIpC will be referred
to as HIF-1α-deficient mice and HIF-1αfl/fl-Mx-Cre- mice treated with pIpC will be
referred to as Control mice.
These mice were used to evaluate the role of HIF-1α in the development of liver
fibrosis. For this study, Control and HIF-1α-deficient mice were subjected to bile duct
ligation. Fourteen days later, markers of cholestasis and liver injury were analyzed.
Bile duct ligation in Control and HIF-1α-deficient mice increased serum levels of ALT,
ALP, and bile acids to a similar extent (Fig. 6).
Effect of HIF-1α α Deletion on Histological Changes in the Liver after Bile
Duct Ligation. Analysis of liver histology indicated no difference between sham-
operated Control and HIF-1α-deficient mice (data not shown). By 14 days after bile
duct ligation, livers of Control mice showed extensive bile duct proliferation and bile
infarcts infiltrated by inflammatory cells (Fig. 7A). Similar changes were observed in bile
duct-ligated HIF-1α-deficient mice (Fig. 7B).
Effect of HIF-1α α Deletion on Bile Duct Proliferation in the Liver after Bile
Duct Ligation. Bile duct epithelial cells were detected in sections of liver by
immunohistochemistry for CK19. CK19 immunostaining was not different between
sham-operated Control and HIF-1α-deficient mice (data not shown). By 14 days after
bile duct ligation, extensive CK19 immunostaining was observed in the livers of Control
mice (Fig. 8A) and HIF-1α-deficient mice (Fig. 8B) after bile duct ligation. Similar
changes were observed in bile duct-ligated HIF-1α-deficient mice (Fig. 8B).
Morphometric analysis revealed that bile duct ligation caused a time-dependent
increase in the area of CK19 staining in the livers of Control mice and HIF-1α-deficient
mice (Fig. 8C). There was no difference between the area of CK19 staining in Control
mice and HIF-1α-deficient mice after bile duct ligation.
Effect of HIF-1α α Deletion on Levels of Type I Collagen and α α-Smooth
Muscle Actin in the Liver after Bile Duct Ligation. After bile duct ligation, activated
hepatic stellate cells and peribiliary fibroblasts synthesize and secrete collagen (21).
15 Download full-text
Accordingly, we determined whether levels of Type I collagen were affected in the livers
of HIF-1α-deficient mice after bile duct ligation. Type Iα1 collagen mRNA (Fig. 9) and
Type I collagen protein (Fig. 10) were significantly increased in the livers of Control and
HIF-1α-deficient mice by 14 days after bile duct ligation. Levels of Type I collagen
mRNA and protein were significantly reduced by approximately 70% and 50%
respectively in HIF-1α-deficient mice after bile duct ligation when compared to Control
mice subjected to bile duct ligation (Figs. 9 and 10).
During the pathogenesis of fibrosis induced by bile duct ligation, hepatic stellate
cells and peribiliary cells differentiate into myofibroblasts and proliferate (21). When this
occurs, levels of α-smooth muscle actin, expressed by these cells, increase. Therefore,
α-smooth muscle actin levels were measured as an indirect indicator of myofibroblast
activation and numbers in liver. α-smooth muscle actin mRNA was significantly
increased in Control and HIF-1α-deficient mice 14 days after bile duct ligation (Fig.
11A). Similar to Type I collagen mRNA levels, levels of α-smooth muscle actin were
significantly lower in HIF-1α-deficient mice when compared to Control mice after bile
duct ligation (Fig. 11A). Immunohistochemical staining for α-smooth muscle actin in
livers of Control mice 14 days after bile duct ligation showed extensive positive α-
smooth muscle actin staining around proliferating bile ducts and within the hepatic
parenchyma in periportal regions (Fig. 11B). Less α-smooth muscle actin
immunostaining was observed in the livers of bile duct-ligated HIF-1α-deficient mice
Effect of HIF-1α α Deletion on Levels of Profibrotic Mediators in the Liver
after Bile Duct Ligation. Levels of PDGF-A, PDGF-B, PAI-1, FGF-2, and CTGF