1Departments of Medicine, 2Biochemistry and Biophysics, and 3Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7032
Submitted 22 January 2003 ; accepted in final form 21 May 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
hepatic inflammation; renin-angiotensin system; cytokines
Recent clinical and experimental evidence suggests that ANG II is involved in the progression of chronic liver diseases. Systemic ANG II is frequently elevated in patients with cirrhosis, and the hepatic renin-angiotensin system is upregulated in both human and rat livers undergoing active fibrogenesis (3, 21). Inhibition of ANG II synthesis and/or the blockade of AT1 receptors markedly attenuate inflammation and extracellular matrix accumulation after chronic liver injury in rats (10, 22, 26, 42, 44). Moreover, patients with chronic hepatitis C and a genetic polymorphism that results in increased synthesis of ANG II develop more severe fibrosis (25). Most importantly, a recent report suggests that the blockade of AT1 receptors has antifibrotic effects in patients with chronic hepatitis C (39).
The mechanisms underlying the pathophysiological effects of ANG II in the
damaged liver are largely unknown. ANG II induces proinflammatory and
profibrogenic effects in hepatic stellate cells (HSCs), a key cell involved in
the hepatic wound-healing response to injury
(2,
4). However, it is unknown
whether increased systemic ANG II induces liver damage and which molecular
mechanisms are involved. In this study, we investigated the effects of
prolonged systemic infusion of ANG II on the normal rat liver. We demonstrate
that infusion of ANG II induces oxidative stress, hepatic inflammation, and
vascular damage, resulting in liver injury. The intracellular pathways
activated by ANG II include NF-B, AP-1, and MAPK activation.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of rat HSCs. HSCs were isolated from adult male Sprague-Dawley rats (>400 g) by in situ perfusion of the livers with collagenase and pronase, followed by arabinogalactan gradient ultracentrifugation, as previously described (32). HSC purity (as estimated by the autofluorescence of the cells by ultraviolet-excited fluorescence microscopy) was >95%. Cells were seeded on uncoated plastic tissue culture dishes and cultured in DMEM (GIBCO-BRL, Grand Island, NY) supplemented with 10% FCS and 2 mM L-glutamine. All animal procedures were performed in accordance with Institutional Animal Care and Use Committee of the University of North Carolina.
Serum biochemical measurements. Blood samples were collected from
all rats at death. Serum alanine aminotransferase, aspartate aminotransferase,
-glutamyltranspeptidase, and bilirubin levels were measured by standard
enzymatic procedures by the Pathology Department, University of North
Carolina. Serum TNF-
and ANG II levels were measured by a sandwich
ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's
instructions.
Measurement of serum endotoxin levels. Blood samples were collected in endotoxin-free vials and centrifuged at 400 g for 15 min at 4°C. Samples were then diluted 1:10 in pyrogen-free water and heated to 75°C for 30 min to remove inhibitors of endotoxin from plasma. The limulus amoebocyte lysate test (Kinetic-QLC; Whittaker Bioproducts, Walkerville, MD) was used for measurements of endotoxin. Samples were incubated at 37°C for 10 min with limulus amoebocyte lysate. The substrate solution was added, and the incubation continued for 20 min. The reaction was stopped with 25% acetic acid. Samples were read spectrophotometrically at 410 nm.
Immunohistochemical studies. Livers were fixed in 10%
phosphate-buffered formalin for 24 h at room temperature, washed twice with
water, stored in 70% ethanol at 4°C, and embedded in paraffin.
Five-micrometer sections were stained with hematoxylin and eosin, Masson
trichrome, and Sirius red. For immunohistochemical analysis, sections were
deparaffinized, rehydrated, and stained by using the DAKO Envision system
(DAKO, Carpinteria, CA) according to the manufacturer's instructions. Briefly,
endogenous peroxidase was blocked with peroxidase-blocking agent and sections
were incubated with anti-4-hydroxynonenal (1:1,000 dilution; Alpha Diagnostic,
San Antonio, TX), anti-smooth muscle -actin (1:1,000; DAKO), anti-CD43
(1:1,000; Serotec, Raleigh, NC), and anti-hypoxia-induced factor
(HIF)-2
(Novus Biologicals, Littleton, CO) for 30 min at room
temperature in PBS containing 1% Tween 20 and 1% bovine serum albumin. Slides
were incubated with peroxidase-linked secondary antibodies for 15 min, washed,
and further incubated with labeled polymer for 10 min at room temperature.
Sections were washed twice with PBS, incubated with 3.3-diaminobenzidine
substrate chromogen for 8 min, washed with water, incubated with
diaminobenzidine enhancer (Innovex Biosciences, Richmond, CA) for 5 min, and
washed with water before being counterstained with hematoxylin. As negative
controls, all specimens were incubated with an isotype-matched control
antibody under identical conditions. The area of positive staining was
measured by using a Macintosh based morphometric analysis system (Apple
Computer, Brea, CA) with NIH Image software (version 1.62).
Hydroxyproline assay. Hydroxyproline content was quantified
colorimetrically in duplicate from 0.2-g liver samples. Tissue was
homogenized in 3 ml of 6 N HCl and hydrolyzed at 110°C for 16 h. The
hydrolysate was filtered, aliquots were evaporated under vacuum, and the
sediment was redissolved in 1.2 ml of 50% isopropanol. The solution was then
incubated with 0.2 ml of 0.84% chloramine T in 42 mM sodium acetate, 2.6 mM
citric acid, and 39.5% (vol/vol) isopropanol (pH 6.0) for 10 min at room
temperature. Next, 0.248 g p-dimethylaminobenzaldehyde, dissolved in
0.27 ml of 60% perchloric acid, and 0.73 ml isopropanol were added and
incubated at 50°C for 90 min. Hydroxyproline content was quantified
photometrically at 558 nm from a standard curve with the amino acid alone and
against a reagent blank. The results were expressed as grams of hydroxyproline
per gram liver.
Western blot analysis. Liver samples were homogenized in lysis
buffer (10 mM HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5%
NP-40, and 25% glycerol) containing protease and phosphatase inhibitors for 30
min at 4°C. After centrifugation, cleared tissue lysates were collected
and stored at -80°C until analysis. Cell lysates were obtained with Triton
lysis buffer as described
(32). Twenty-five micrograms
were loaded onto 10% SDS-acrylamide gels and blotted onto nitro-cellulose
membranes. Membranes were incubated in blocking buffer containing antibodies
against phospho-ERK (Cell Signaling, Beverly, MA), cyclooxygenase-2 (COX-2),
inducible nitric oxide synthase (iNOS), phospho-c-Jun and -tubulin
(Santa Cruz Biotechnology, Santa Cruz, CA), and smooth muscle
-actin.
After extensive washing, the membranes were incubated with blocking buffer
containing horseradish peroxidase-conjugated antibodies (Santa Cruz
Biotechnology) at a dilution of 1:1,000. Proteins were detected by enhanced
chemoluminescence (Amersham, Arlington Heights, IL). For detection of collagen
I secretion, cell media were precipitated with sodium sulfite and resuspended
in acetic acid as described
(37). After being blotted,
membranes were probed with anti-human collagen type I antibody (1:1,000;
Biodesign, Saco, ME).
Electrophoretic mobility shift assay. Fresh liver tissue samples
were homogenized in buffer A (in mM: 10 HEPES, pH 7.9, 10 KCl, 1.5
MgCl2, and 0.5 DTT) containing protease and phosphatase inhibitors,
incubated for 30 min at 4°C, and then lysed in 10% NP-40. After
centrifugation, nuclei were lysed in buffer C (10 mM HEPES, 25%
glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, and 0.5% NP-40)
containing protease and phosphatase inhibitors. Eight micrograms of nuclear
proteins were incubated with 100 pg of a 32P-labeled probe
containing the AP-1 (5'-GTAAAGCATGAGTCAGACACCTC-3') or NF-B
(top: 5'-GCAGAGGGGACTTTCCGAGA-3'; bottom:
5'-GTCTCGGAAAGTCCCCTCTG-3') consensus sites in buffer containing
(in mM) 10 HEPES, 2 MgCl2, 50 KCl, 1 DTT, 0.1 EDTA and 20% glycerol
in the presence of single-stranded oligonucleotide (25 µg/ml) and
poly(dI/dC) (25 µg/ml) for 20 min at room temperature
(13). For competition assay,
one sample was incubated with 10 ng unlabeled probe. Complexes were separated
by electrophoresis on nondenaturing 4% acrylamide gels and assayed by
autoradiography and PhosphorImager analysis (Molecular Dynamics, Sunnyvale,
CA).
ELISA for IL-1 and TNF-
. Liver tissue
samples were homogenized in lysis buffer containing 25 mM HEPES, 0.1% CHAPS, 5
mM MgCl2, 1.3 mM EDTA, 1 mM EGTA, and phosphatase and protease
inhibitors for 30 min at 4°C. After centrifugation, cleared tissue lysates
were collected and stored at -80°C. A sandwich ELISA for rat IL-1
and TNF-
(R&D Systems, Minneapolis, MN) was performed by using 1:5
dilutions according to the manufacturer's instructions.
NF-B-responsive luciferase assay. Rat HSCs were
infected with a recombinant adenoviral vector expressing a luciferase reporter
gene driven by NF-
B transcriptional activation (Ad5NF-
BLuc) for
12 h as described (20). Medium
was replaced, and cells were stimulated with ANG II
(10-8 M) for 8 h. NF-
B-mediated transcriptional
induction was assessed by the luciferase assay system (BD Pharmingen, San
Diego, CA). Luciferase activity (relative light units) was normalized to the
protein concentration.
Statistical analysis. Results are expressed as means ± SE. Significance was established by using the Mann-Whitney U-test. Differences were considered significant if P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Effect of ANG II infusion on serum liver enzymes and endotoxin
levels. ANG II infusion resulted in increased ANG II serum concentration
(0.3 ± 0.03, 1.2 ± 0.2, and 1.8 ± 0.5 ng/ml in controls
and rats receiving 15 and 50
ng·kg-1·min-1 ANG
II, respectively; P < 0.01 vs. saline). ANG II infusion increased
alanine aminotransferase, aspartate aminotransferase, and
-glutamyltranspeptidase serum levels in a concentration-dependent
manner, whereas bilirubin levels remained unchanged
(Fig. 2). ANG II-infused rats
showed higher serum TNF-
levels compared with control rats (3.3
± 1.3, 8.4 ± 2.1, and 9.3 ± 3.1 in controls and rats
receiving 15 and 50
ng·kg-1·min-1 ANG
II, respectively; P < 0.05 vs saline). Finally, ANG II-infused
rats showed higher serum endotoxin levels compared with control rats (8.7
± 0.8, 23.6 ± 3.4, and 37.8 ± 5.5 pg/ml in controls and
rats receiving 15 and 50
ng·kg-1·min-1ANG II,
respectively; P < 0.01 vs saline).
|
Histological changes in livers from ANG II-infused rats. Histological examination of ANG II (15 ng·kg-1·min-1)-infused livers showed preserved hepatic parenchyma with no apparent hepatocyte damage. Thirty percent of rats receiving ANG II at pressor doses (50 ng·kg-1·min-1) showed signs of mild ischemic hepatitis with necrosis in pericentral areas (not shown). Infiltration of mononuclear cells and thickening of the limiting membrane were observed in portal tracts from ANG II-treated rats (Figs. 3A and 4). Proliferation of bile ducts was observed in 30% of portal tracts in ANG II-treated rats (not shown). Hepatic small vessels showed signs of vasculitis and wall thickening with frequent vascular thrombosis (Fig. 4). As expected, kidneys from ANG II-treated rats show profound inflammation and collagen deposition (not shown). Similarly, the abdominal aorta showed infiltration of mononuclear cells and marked wall thickening (Fig. 3B). These latter changes were more pronounced in rat infused with the pressor doses of ANG II.
|
|
Immunostaining studies. Because ANG II induces proliferation of
culture-activated HSCs (2), we
first examined whether ANG II infusion induces the accumulation of smooth
muscle -actin-positive cells, a marker of activated HSCs, in the liver.
Infusion of ANG II induced a marked increase in the number of smooth muscle
-actin-positive stellate cells, mainly observed in the pericentral area
(0.1 ± 0.02, 8.2 ± 1.1, and 12.1 ± 2.2 positive cells per
field in rats infused with saline and with 15 and 50
ng·kg-1·min-1 ANG
II, respectively; P < 0.01 vs saline;
Fig. 5A). To
investigate the infiltration of inflammatory cells in the liver parenchyma,
CD43-positive cells were counted. CD43 is typically expressed by infiltrating
mononuclear cells and lymphocytes
(29). ANG II perfusion induced
the infiltration of CD43-positive cells in the hepatic parenchyma, which were
mainly observed at the pericentral areas (2.2 ± 0.1, 11.2 ± 2.6,
and 14.1 ± 3.5 positive cells per field in rats infused with saline and
with 15 and 50
ng·kg-1·min-1 ANG
II, respectively; P < 0.05 vs. saline;
Fig. 5B). ANG II
infusion also induced tissue hypoxia, as demonstrated by increased expression
of HIF-2
in the hepatocytes located in the pericentral areas
(Fig. 5C). Finally,
because oxidative stress mediates the effects of ANG II in other tissues, we
assessed whether ANG II infusion induces oxidative stress in the rat liver.
ANG II-infused livers showed a marked increase in lipid peroxidation products,
as demonstrated by intense staining for 4-hydroxynonenal-modified proteins
(Fig. 5D). These
results indicate that systemic infusion of ANG II induces activation of HSCs,
infiltration of inflammatory cells, and oxidative stress in the rat liver.
|
Effect of ANG II perfusion on hepatic proinflammatory proteins. To
confirm that ANG II exerts inflammatory actions on the rat liver, we measured
the expression of cytokines and other proinflammatory proteins in the rat
liver. ANG II infusion increased the hepatic concentration of the inflammatory
cytokines TNF- and IL-1
compared with livers infused with saline
(Fig. 6, A and
B). Similarly, ANG II infusion induced the expression of
typical proinflammatory proteins such as COX-2 and iNOS, as assessed by
Western blotting (Fig.
6C). These results further demonstrate that ANG II
infusion induces inflammatory events in the rat liver.
|
Effect of ANG II perfusion on collagen deposition. Because blockade of ANG II's biological actions ameliorates collagen deposition in experimental liver fibrosis (26, 39, 42, 44), we explored whether increased systemic ANG II induces collagen accumulation in rat liver. ANG II infusion did not significantly increase collagen content in the hepatic parenchyma, as assessed by Sirius red staining. However, increased Sirius red staining was observed in the small portal vein and arterial vessel walls, limiting membrane of portal tracts, and central veins (Fig. 7, A and B). Hepatic collagen was also assessed by measuring hepatic hydroxyproline content. Only pressor doses of ANG II induced a slight increase in hydroxyproline content, whereas infusion of ANG II at subpressor doses had no effect (Fig. 7C). These results suggest that infusion of ANG II to normal rats induces thickening and fibrosis of small hepatic vessels but no parenchymal fibrosis.
|
Intracellular pathways stimulated by ANG II in the rat liver. We
also explored whether ANG II infusion stimulates redox-sensitive intracellular
pathways, including MAPK and the transcription factors AP-1 and NF-B.
ANG II infusion increased phosphorylation of c-Jun (a specific substrate of
JNK) and ERK-2 by six- and fivefold, respectively, in the rat liver compared
with saline-infused rats, as assessed by Western blot analysis
(Fig. 8A). Moreover,
ANG II increased DNA-binding activity of the transcription factors AP-1 and
NF-
B by four- and threefold, respectively, compared with control rats
(Fig. 8B).
|
ANG II accelerates the activation of primary rat cultured HSCs. To
test whether ANG II induces activation of HSCs in vitro, early-cultured rat
HSCs were incubated for 2 days in the presence or absence of ANG II
(10-8 M). As shown in
Fig. 9, ANG II increased the
expression of smooth muscle -actin and increased the secretion of
collagen I to the culture medium. Moreover, ANG II (10-8
M) induced the activation of the transcription factor NF-
B, as assessed
by luciferase-driven NF-
B assay. All of these effects were prevented by
the AT1 receptor antagonist losartan (10-7
M).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our experimental model consisting of infusing ANG II through a subcutaneous
pump into rats has been used by many investigators
(1,
11,
15,
29,
35). In the current study, we
used both subpressor and pressor doses of ANG II. Liver injury was more severe
when pressor doses of ANG II were used. Although renal function was not
measured in the current study, the histological changes found in the kidneys
of ANG II-infused rats strongly suggest that renal function is reduced in
these animals. However, because of the abnormal liver histology that we
observed, it is unlikely that an ANG II-induced rise in serum liver enzymes is
solely due to impaired renal function. Importantly, the pathogenic effects
were also observed when ANG II was infused at subpressor doses, indicating
that these effects are not dependent on an increase in arterial pressure. ANG
II infusion caused inflammation and sclerosis of small vessels and thrombosis.
These effects may be influenced by hepatic hypoxia, since ANG II infusion
induced HIF-2 in the liver. Moreover, a marked oxidative stress was
found, as assessed by increased lipid peroxidation of protein adducts. Similar
in vivo effects have been previously described in the heart, the vasculature,
and the kidney (1,
29,
35,
43). In these organs, ANG II
induces infiltration by mononuclear cells and progressive fibrosis. However,
the severity of histological damage observed in the kidneys was more
pronounced than that found in the liver, suggesting that the kidneys are more
sensitive to increased systemic ANG II levels. The hepatic effects induced by
prolonged infusion of ANG II can be due either to local or remote effects. The
finding that the aorta and kidneys show profound inflammation strongly
indicates that ANG II infusion induces pathological effects in other organs
than the liver. A potential mechanism is that ANG II infusion increases gut
permeability, leading to gram-negative bacterial translocation. Increased
bacterial products in serum would cause hepatic inflammation in the context of
a generalized inflammatory response. The finding that ANG II infusion markedly
increases endotoxin serum levels supports this hypothesis. Alternatively, ANG
II could induce proinflammatory effects in the liver by causing direct
stimulation of hepatic cells. ANG II induces proinflammatory effects on HSCs
(4). Whether ANG II induces
similar effects in other cell types involved in liver inflammation (i.e.,
Kupffer cells) is unknown.
An important finding of our study is that ANG II induces activation of HSCs in vivo, a key event in liver fibrogenesis. Following activation, HSCs secrete abundant extracellular matrix and a number of proinflammatory cytokines (27). We also show that ANG II accelerates the activation process of HSCs in culture. Collectively, these results indicate that ANG II is a powerful agonist for these cells. However, although hepatic inflammation was commonly observed in ANG II-treated rats, no extensive parenchymal fibrosis was found. Several reasons can explain this finding. First, it is conceivable that systemic ANG II only increases collagen synthesis when the liver is being damaged by another agent (i.e., alcohol, viral infection). Second, a local renin-angiotensin system has been recently described in both rat and human liver (21). This system is upregulated in damaged livers, suggesting that locally synthesized ANG II could participate in hepatic fibro-genesis. Whether the local, rather than systemic, renin-angiotensin system induces collagen synthesis in the damaged liver is unknown and deserves further investigation.
We provide in vivo evidence that ANG II activates the transcription factors
NF-B and AP-1 and stimulates the intracellular signaling molecules ERK
and c-Jun in the rat liver. Comparable results have been described in the rat
kidney, suggesting that ANG II infusion stimulates similar intracellular
pathways in different organs. NF-
B activation mediates the inflammatory
actions of ANG II in many tissues and is activated in a number of human and
experimental liver diseases
(4-6,
24,
29). Importantly, inhibition
of NF-
B activation prevents experimental liver injury
(41). In the current study,
NF-
B-dependent proteins such as COX-2 and iNOS, which are typically
involved in inflammatory processes, were upregulated in the ANG II-infused
livers. Whether NF-
B inhibition attenuates the effects of ANG II in the
liver is unknown. We also found increased AP-1 binding activity in the ANG
II-treated livers. AP-1 mediates proinflammatory and profibrogenic actions in
HSCs (16). However, its
expression during liver fibrogenesis in vivo is unknown. Finally, ANG II
stimulates the MAPKs JNK and ERK in the rat liver. JNK modulates fibrogenic
and inflammatory actions in HSCs and is activated after liver injury
(32,
34). Importantly, JNK
inhibitors have protective effects in ischemia-reperfusion and liver
transplantation in rats (40).
ERK is induced after acute liver injury and regulates proliferation and
biological actions in many liver cell types
(17).
Because of the in vivo nature of our study, it is difficult to ascertain
which specific cell type mediates the hepatic effects induced by ANG II
infusion. Several cell types are potential candidates. Although ANG II induces
activation and stimulates NF-B in rat primary HSCs, other cell types
such as hepatocytes and Kupffer cells are potential targets for ANG II.
Whether ANG II exerts proinflammatory effects in these cells has not been
addressed in the current study and deserves further investigation. Moreover,
the relative importance of the different intracellular signaling pathways
induced by ANG II infusion (ERK, NF-
B, c-Jun/AP-1) in the pathogenesis
of liver inflammation is uncertain.
The results of this study may have important pathophysiological implications. In particular, several of the effects observed after ANG II infusion could contribute to the progression of chronic liver diseases. First, oxidative stress plays an important role in the hepatic fibrogenic response to different etiologic agents. Increased reactive oxygen species and resulting lipid peroxidation products are commonly detected in livers from patients with alcohol abuse, hepatitis C virus infection, iron overload, or chronic cholestasis, as well as in most types of experimental liver fibrogenesis (23). Moreover, antioxidant agents attenuate the development of hepatic fibrosis in rodents and exert beneficial effects in patients with chronic liver diseases (12). Second, ANG II infusion induces vascular thrombosis in the liver. The procoagulant effect of ANG II has been previously reported in other organs (38). Thrombosis of small vessels is frequently found in chronic liver diseases, including alcoholic hepatitis, hepatic rejection, and liver cirrhosis (8, 19). Vascular thrombosis can impair liver perfusion and has been implicated in the pathogenesis of portal hypertension and fibrosis progression (19). Potential mechanisms include impaired liver oxygenation and inflammatory effects of coagulation proteins. Therefore, the procoagulant effect of ANG II may contribute to liver damage. Finally, systemic ANG II could contribute to the increased intrahepatic vascular resistance found in chronic liver diseases. Circulating ANG II levels are markedly increased in patients with cirrhosis and portal hypertension, and ANG II inhibitors decrease portal pressure (10, 33). We previously reported that ANG II increases intracellular Ca2+ concentration and cell contraction in activated, but not quiescent, HSCs (2). The finding that ANG II induces activation of resident HSCs reinforces the hypothesis that ANG II may play a role in the pathogenesis of portal hypertension.
In summary, we provide evidence that prolonged systemic infusion of ANG II
to normal rats induces liver damage consisting of areas of necrosis,
vasculitis with thrombogenic events, oxidative stress, and activation of HSCs.
Moreover, we demonstrate that ANG II induces NF-B and AP-1 activity in
the liver, as well as MAPK activation. These results shed light on the
mechanisms involved in the protective effect of ANG II inhibition in chronic
liver diseases.
![]() |
DISCLOSURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|