1Department of Internal Medicine and Liver Center, University of Alabama at Birmingham, and 2Birmingham Veterans Administration Medical Center, Birmingham, Alabama, 35294
Submitted 16 July 2003 ; accepted in final form 11 September 2003
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ABSTRACT |
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partial portal vein ligation; common bile duct ligation; thioacetamide; nitric oxide synthase; endothelin B receptor
Chronic common bile duct ligation (CBDL) in the rat is the only recognized model system for the study of HPS (10, 11). In this model, biliary cirrhosis is associated with an early increase in pulmonary endothelial NO synthase (eNOS) levels followed by increased inducible NOS (iNOS) and heme oxygenase-1 (HO-1) expression in accumulated pulmonary intravascular macrophages. These alterations lead to the development and increasing severity of intrapulmonary vasodilatation and gas exchange abnormalities analogous to human HPS (3, 10, 11). Increased hepatic production and plasma levels of endothelin-1 (ET-1) accompanied by pulmonary vascular ETB receptor overexpression are seen early after the onset of CBDL and appear to trigger increases in pulmonary eNOS levels and the onset of HPS (27, 28, 50). Bacterial translocation, leading to TNF--mediated increases in pulmonary iNOS and HO-1 expression, is also an important factor in the development of vasodilatation (37). Prehepatic portal hypertension induced by partial portal vein ligation (PVL) is used as a control model, because these animals develop a similar degree of portal hypertension and hyperdynamic circulation to CBDL animals but do not develop HPS (11).
Whether biliary cirrhosis is unique in stimulating hepatic production and release of ET-1 and initiating the onset of HPS is undefined. Specifically, no studies have evaluated whether established models of hepatocellular injury leading to cirrhosis may also result in experimental HPS and how plasma TNF- levels, the degree of portal hypertension, and ET-1 production relate to the molecular and physiological alterations of HPS. The two most widely used rodent models of toxic hepatocellular injury leading to cirrhosis are induced by chronic administration of either carbon tetrachloride (13, 35) or thioacetamide (TAA) (15, 17, 44). Carbon tetrachloride administration has been associated with direct pulmonary toxicity including fibrosis (13, 35), whereas TAA administration has not. Therefore, the present study was undertaken to assess the role of ET-1 in the molecular and physiological changes associated with experimental HPS by evaluating partial PVL, biliary, and TAA-induced cirrhosis.
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MATERIALS AND METHODS |
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Arterial blood gas analysis. Arterial blood was drawn from the femoral artery as described (11) via extension tubing to ensure that values reflected resting arterial gas exchange. Arterial blood gas analysis was performed on an ABL 520 radiometer (Radiometer America, Westlake, OH) in the Clinical Laboratory, University of Alabama at Birmingham Hospital (Birmingham, AL). The alveolar-arterial oxygen gradient was calculated as 150 - (PaCO2/0.8) - PaO2.
Microsphere protocol. The pulmonary microcirculation was evaluated by using an established technique (11). Cross-linked (2.5 x 106) colored polystyrene-divinylbenzene microspheres (size range, 5.5-10 µm; Interactive Medical Technologies, Irvine, CA) were injected through a femoral vein catheter, after removing an aliquot of microspheres, to verify the numbers and sizes of microspheres injected. A blood sample withdrawn from a femoral arterial catheter beginning at the time of femoral vein injection measured microspheres passing through the lung microcirculation. Numbers and sizes of microspheres were assessed by using a Leitz Laborlux microscope (Wetzlar, Germany) with a color video imaging and digital analysis system (Image Pro 5.0; Media Cybernetics, Silver Spring, MD) and counted directly. Total numbers of microspheres passing through the microcirculation were calculated as arterial blood sample microspheres/milliliter x estimated blood volume (24). Intrapulmonary shunting was calculated as an intrapulmonary shunt fraction (%) = (total numbers of microspheres passing through the pulmonary microcirculation/total microspheres injected into the venous circulation) x 100.
Determination of plasma TNF- and endotoxin concentrations. Plasma TNF-
levels were measured with a commercially available solid-phase sandwich enzyme-linked immunosorbent assay according to the protocol supplied by the manufacturer (R&D Systems, Minneapolis, MN). Standards and samples were incubated with a TNF-
antibody-coated 96-well microtiter plate. An enzyme-linked polyclonal antibody specific for rat TNF-
was then added after washing. The intensity of the color was measured in a microplate reader (Molecular Devices, Sunnyvale, CA).
Endotoxin concentrations were measured by a Limulus amebocyte lysate test. Plasma samples were serially diluted with sterile endotoxin-free water and heat-treated to destroy inhibitors that can interfere with activation. Endotoxin content was determined as described by the manufacturer (QCL-1000 kit; Bio Whittaker, Walkersville, MD). Endotoxin standards were tested in each run and the concentration of endotoxin in the test samples was calculated by comparison with the standard curve.
ET-1 radioimmunoassay. Plasma and tissue ET-1 concentrations were measured via radioimmunoassay (RIA; Phoenix Pharmaceuticals, Mountain View, CA). Extraction of ET-1 from plasma was accomplished by acidification and elution over Sep-Pak C18 columns (Waters, Milford, MA). Liver and lung tissues were homogenized and prepared as previously described (27). Samples were reconstituted in RIA buffer and subjected to RIA with the use of a rabbit ET-1 antiserum. Recovery from the Sep-Pak C18 columns averaged 90%, and the sensitivity of the assay for ET-1 was 1.5-2.0 pg.
Western blot analysis. eNOS, ETB receptor, iNOS, HO-1, or ED1 protein levels were measured in lung as described (10, 27, 28). Equal concentrations of protein from lung were fractionated on Tris·HCl-ready gels (Bio-Rad Laboratories, Hercules, CA) and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). Incubation with antibodies to eNOS (Transduction Laboratories and Pharmingen, Lexington, KY), ETB receptor (Calbiochem-Novabiochem, San Diego, CA), iNOS (Transduction Laboratories and Pharmingen), HO-1 (StressGen Biotechnologies, Victoria, BC, Canada), or ED1 (Serotec, Raleigh, NC), a general macrophage and monocyte marker, was followed by the addition of horseradish peroxidase-conjugated secondary antibodies and detection with enhanced chemiluminescence (Amersham). Autoradiographic signals were assessed by using an Astra scanner (UMAX, Fremont, CA) and quantitated with ImagePC software (Scion, Frederick, MA). Signal intensity was shown to be a linear function of sample concentration over the range analyzed.
Liver histology and lung immunohistochemistry. Liver samples were fixed in 10% neutral buffered formalin solution, and lung samples were fixed in 4% paraformaldehyde. Paraffin-embedded tissues were sectioned at 5 µm. Liver sections were stained with Masson's trichrome stain (14). Lung staining was performed as described (32). After preparation and blocking, sections were incubated with ETB receptor (Calbiochem-Novabiochem), iNOS (Transduction Laboratories and Pharmingen) or ED1 (Serotec) antibodies; washed; and incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA). After peroxidase-labeled streptavidin (Signet Laboratories, Dedham, MA) and diaminobenzidine (Biogenex, San Ramon, CA) development, sections were photographed by using an axiophot microscope (Nikon, Melville, NY). Control sections were incubated with secondary antibody alone.
Statistics. Data were analyzed by using Student's t-test or ANOVA with Bonferroni correction for multiple comparisons between groups. Measurements are expressed as means ± SE. Statistical significance was designated as P < 0.05.
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RESULTS |
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Arterial blood gases and pulmonary microvascular evaluation (Table 1) revealed a progressive increase in the alveolar-arterial oxygen gradient and significant intrapulmonary shunting in CBDL animals, but not PVL animals, indicating the development of HPS after CBDL as observed previously (10, 11). There was a trend toward an increase in the alveolar-arterial oxygen gradient within 2 wk after TAA treatment related to a mild respiratory alkalosis although this did not reach statistical significance. This was not accompanied by a decline in the arterial PO2 or an increase in the alveolar-arterial oxygen gradient to >15 mmHg in contrast to findings in CBDL and human HPS (10, 11, 40). Microsphere analysis confirmed that the passage of sized microspheres through the pulmonary microcirculation was unchanged in 2- and 8-wk TAA-treated animals compared with controls, indicating a lack of vasodilatation.
Evaluation of the endothelin system and plasma TNF- and endotoxin levels in prehepatic portal hypertension and biliary and nonbiliary cirrhosis. To evaluate ET-1 production and pulmonary vascular ETB receptor expression and plasma TNF-
and endotoxin levels in relation with development of experimental HPS, we measured these factors in our models (Table 2, Figs. 2 and 3). Hepatic ET-1 production and plasma ET-1 levels rose progressively after CBDL and correlated with the onset and progression of HPS as seen previously (27). In contrast, hepatic ET-1 production and plasma ET-1 levels did not rise after PVL or TAA treatment and lung ET-1 levels were not altered in any model. Pulmonary ETB receptor levels increased significantly in all models as portal hypertension increased, and staining was localized to the pulmonary endothelium (28). There was a progressive increase in plasma TNF-
levels in both CBDL and TAA-treated animals not observed in PVL animals. TNF-
levels were significantly higher in TAA-treated animals than in CBDL animals despite the lack of development of HPS in nonbiliary cirrhosis. Plasma endotoxin levels were measured to assess the contribution of portosystemic shunting and bacterial translocation to TNF-
production. Levels increased significantly after CBDL as TNF-
levels increased but did not rise in either PVL or TAA-treated animals despite the marked increase in TNF-
levels after TAA treatment.
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Pulmonary eNOS, iNOS, HO-1, and ED1 levels in prehepatic portal hypertension and biliary and nonbiliary cirrhosis. To assess the levels and distribution of proteins that produce vasoactive mediators implicated in the development and progression of experimental HPS in relation with ET-1 and TNF- alterations, we measured eNOS, iNOS, HO-1, and ED1 levels in lung homogenates and performed immunohistochemical analysis (Figs. 4 and 5). There was a progressive increase in pulmonary eNOS levels after CBDL and a dramatic rise in HO-1 expression at 5 wk associated with the onset and progression of HPS as previously described (3). The eNOS increase was localized to the pulmonary vascular endothelium, and the HO-1 increase occurred in pulmonary intravascular macrophages that accumulated progressively after CBDL, as evidenced by a significant increase in ED1 levels, in line with prior results (3, 10). In contrast, eNOS and HO-1 levels did not increase significantly in PVL or TAA-treated animals. However, there was accumulation of pulmonary intravascular macrophages in TAA lung, to a lesser degree than CBDL, confirmed by both an increase in pulmonary ED1 levels and immunohistochemistry. iNOS levels were increased modestly in these cells in 8-wk TAA-treated animals but were not associated with the development of HPS.
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Effects of exogenous ET-1 infusion on the development of HPS in PVL animals. To define how ET-1 influences the development of molecular and physiological changes associated with HPS in vivo, we infused ET-1 intravenously at doses similar to those seen in the plasma of CBDL animals for 2 wk to normal and PVL animals (Table 3, Fig. 6). ET-1 infusion was associated with an increase in pulmonary vascular eNOS levels and the development of HPS in PVL animals in which pulmonary vascular ETB receptor expression is increased but not in normal animals as described previously (28, 50). In addition, ET-1 infusion also increased plasma TNF- levels in PVL animals without altering portal or systemic pressures or resulting in endotoxemia. These changes were associated with a significant increase in intravascular macrophage accumulation in PVL animals but not with a rise in pulmonary iNOS or HO-1 levels. ET-1 infusion did not significantly alter plasma TNF-
, endotoxin levels, or pulmonary iNOS, HO-1, or ETB receptor levels in normal animals.
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DISCUSSION |
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Alterations in the endothelin system are well documented in liver disease. Specifically, hepatic production (27, 36, 39) and circulating levels (1, 27, 31) of ET-1 have been observed in some forms of experimental and human cirrhosis. CBDL appears to be unique in that hepatic ET-1 production is increased within 7 days after ligation, before the onset of portal hypertension, whereas ET-1 increases in humans have been detected only in advanced disease (1, 31). In CBDL, the rise in ET-1 is likely related to production in both stellate cell and biliary epithelium (39). In addition, biliary levels of ET-1 are high in rodents both under normal conditions and after bile duct ligation (26) suggesting that retrograde release of ET-1 from bile into blood may be a unique consequence of biliary obstruction. The present study supports that bile duct obstruction is one important contributor to hepatic and plasma ET-1 levels by showing that toxic hepatocellular injury without bile duct obstruction fails to significantly increase hepatic or plasma ET-1 despite the development of cirrhosis and portal hypertension. Our finding that plasma ET-1 levels do not rise in TAA cirrhosis is in agreement with recent work (48).
We have also found that pulmonary vascular endothelial ETB receptor expression is increased in the lung in TAA-treated animals as portal hypertension increases. This finding is consistent with previously documented changes in prehepatic portal hypertension and in biliary cirrhosis (28). Although two vascular ETB receptor types have been identified, one in endothelial cells that upregulates eNOS activity and expression and produces NO and the other in smooth muscle cells functioning similar to the vasoconstrictive ETA receptor (29, 46), our present and prior immunohistochemical results support the finding that the major increase in our models occurs in the endothelium (28). This finding is consistent with the observation that ET-1 causes an ETB receptor-dependent increase in eNOS levels and NO production in short-term cultured pulmonary artery segments from PVL and CBDL animals relative to control (28). Our findings also support the concept that the development of portal hypertension is an important factor in increased lung endothelial ETB receptor expression and in susceptibility to ET-1-mediated vascular alterations. Although the mechanisms have not been directly explored in vivo, ETB receptor expression can be altered by a number of factors including changes in flow (2, 30, 38) and cytokine production (25, 33) both of which occur in the setting of portal hypertension.
Molecular changes associated with the development of experimental HPS include an increase in pulmonary vascular eNOS expression in the lung beginning within 2 wk after CBDL, which has been attributed to circulating ET-1 acting through a selective increase in pulmonary endothelial ETB receptors in the setting of portal hypertension (26-28, 50). In addition, increased iNOS and HO-1 expression in accumulated intravascular macrophages also occurs and appears dependent on TNF- overproduction related to bacterial translocation (3, 37). Our findings support the importance of ET-1 in triggering eNOS expression in the lung in the setting of portal hypertension by documenting that eNOS changes do not occur in TAA cirrhosis or after PVL in which circulating ET-1 levels are not increased and by confirming that exogenous ET-1 increases pulmonary eNOS levels and the development of HPS in PVL but not normal animals. Increase in plasma ET-1 levels after ET-1 infusion in PVL animals is smaller than that seen in 2-wk CBDL animals but significant relative to saline-infused PVL animals. The finding that small changes in luminal ET-1 concentrations can influence pulmonary vascular tone is not surprising because the release of small quantities of endothelial ET-1 are recognized to stimulate ETB-mediated NO production through an autocrine loop (16, 29). In addition, the lung vasculature is the major site of endothelin clearance, an event that occurs through the high-affinity ETB receptor (12). Therefore, in the setting of increased pulmonary endothelial ETB receptor levels in portal hypertension, small changes in ET-1 levels are likely to have significant effects on eNOS. In addition to effects on pulmonary eNOS, we also find that circulating ET-1 modulates plasma TNF-
levels and intravascular macrophage accumulation in the lung in PVL animals, a finding consistent with the ability of ET-1 to influence TNF-
expression and monocyte adherence in vitro (5, 43). The observation that our low concentration ET-1 infusion did not alter systemic or portal pressures and did not influence circulating endotoxin levels supports that effects were not due to splanchnic vasoconstriction modulating intestinal permeability. Together, these findings suggest that ET-1 influences both pulmonary microvascular eNOS, circulating TNF-
, and intravascular macrophage accumulation in the lung in HPS.
Our results also show that TNF- overproduction occurs in both biliary and nonbiliary cirrhosis. The finding that both endotoxin and TNF-
levels are elevated in 5-wk CBDL animals is in agreement with the established concept that increased TNF-
levels, driven both by bacterial translocation and decreased hepatic clearance, occur in advanced experimental cirrhosis (49). The increase in circulating TNF-
in these models plays an important role in the maintenance of the hyperdynamic circulatory state by modulating splanchnic and systemic vascular eNOS activity (49) and could regulate pulmonary vascular eNOS production in HPS. Our finding that TNF-
levels are more than sixfold higher in both 2- and 8-wk TAA animals than in CBDL animals and are not associated with measurable changes in serum endotoxin levels or biochemical evidence of advanced liver disease supports the concept that decreased hepatic clearance and bacterial translocation are unlikely to be a major source of TNF-
in this model. Increased production in mononuclear cells that have been described to accumulate in the liver after TAA treatment (34) or in mesenteric lymph nodes after intraperitoneal injection of TAA are other potential sources. The dramatic rise in TNF-
levels after TAA treatment is not associated with pulmonary eNOS alterations or with the development of HPS, although it is associated with pulmonary intravascular macrophage accumulation and iNOS overproduction. These findings agree with the recent concept that TNF-
overproduction contributes to pulmonary intravascular macrophage accumulation and HPS after CBDL but support that TNF-
alone is insufficient for the full development of the syndrome (37). These results also suggest that the cell type of presumed NO overproduction in the pulmonary vasculature may influence effects on vascular tone. One mechanism for this effect may relate to an increase in cyclooxygenase-2 activity and vasoactive prostanoid production observed in activated macrophages, an effect mediated in part by interactions between NO and superoxides generated after activation (21). Increased pulmonary levels of the potent vasoconstrictor thromboxane A2 attributed to production in intravascular macrophages have been found in CBDL animals (4). Production of such vasoconstrictors in macrophages could counteract NO-mediated vasodilatation in TAA lung, particularly because endothelial NO production is not increased in this situation. However, understanding why increased iNOS production in intravascular macrophages alone does not cause pulmonary vasodilatation requires further evaluation.
One hypothesis, on the basis of our observation that both ET-1 and TNF- levels are increased in experimental HPS, is that these mediators interact to influence the development of pulmonary vascular abnormalities. Our finding that exogenous administration of ET-1 to PVL animals influences both circulating TNF-
levels and pulmonary intravascular macrophage accumulation during the development of HPS is consistent with this concept and with a growing body of evidence. ET-1 has been shown to modify monocyte adhesion by both direct effects on monocytes as well as by modulation of cell adhesion molecule and macrophage chemokine expression and NO production in endothelial cells (5, 18, 20, 23). In addition, ET-1 can increase TNF-
production by macrophages (43). TNF-
also modulates monocyte adhesion directly and influences cell adhesion molecule expression and can influence ETB receptor expression and NO production in endothelial cells (19, 47, 49). These findings support that ET-1 and TNF-
interactions, possibly occurring both locally in the lung vasculature and in other vascular beds and organs, contribute to the development of experimental HPS. Defining the cellular sites and mechanisms of these interactions in experimental HPS may provide insight into the pathogenesis of human disease and allow targeting of new therapies.
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ACKNOWLEDGMENTS |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02030 and a Veterans Affairs Merit Review Grant (to M. B. Fallon).
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FOOTNOTES |
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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.
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REFERENCES |
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