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The hepatopulmonary syndrome (HPS) is characterized by intrapulmonary vascular dilatations and an increased alveolar-arterial oxygen difference (AaPO2). Exhaled nitric oxide (NO) concentrations are elevated, suggesting that pulmonary NO overproduction may be the mechanism underlying HPS. We investigated whether common bile duct ligation in rats results in lung NO overproduction and whether normalization of NO synthesis by a 6-wk course of
NG-nitro-L-arginine methyl ester (L-NAME) (5 mg · kg
1 · d1) prevents HPS. Untreated cirrhotic rats showed increases in AaPO2 and
in cerebral uptake of intravenous 99mTc-labeled albumin macroaggregates (indicating intrapulmonary vascular dilatations), with decreases in pulmonary vascular resistance and in pulmonary vasoconstriction induced by angiotensin II and hypoxia. Increases were
found in exhaled NO; pulmonary total and calcium-dependent NO
synthase (NOS) activities; and pulmonary expression of inducible
and, to a lesser extent, endothelial NOS. Accumulation of intravascular macrophages accounted for the inducible NOS expression.
L-NAME normalized AaPO2, brain radioactivity, pulmonary vascular resistance, reactivity to hypoxia and angiotensin II, exhaled NO, and
NOS activities. These findings suggest that HPS and the associated
reduced response to pulmonary vasoconstrictors seen in untreated
cirrhotic rats are related to increased pulmonary NO production dependent primarily on increases in the expression and activities of
inducible NOS within pulmonary intravascular macrophages.
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Keywords: hepatopulmonary syndrome; nitric oxide; nitric oxide synthase; macrophage
Hepatopulmonary syndrome (HPS) is characterized by intrapulmonary vascular dilatations, an increased alveolo-arterial oxygen difference (AaPO2), and liver disease or portal hypertension, in the absence of other cardiopulmonary diseases (1- 3). HPS occurs in approximately 20% of patients with chronic liver disease or portal hypertension (1). Rats with cirrhosis induced by common bile duct ligation (4, 5) develop HPS with arterial gas exchange abnormalities, intrapulmonary vascular dilatations, and reduced responses to pulmonary vasoconstrictors. This model replicates the abnormalities seen in human HPS (4, 6, 7).
Recent studies found that exhaled nitric oxide (NO) concentrations were higher in patients with cirrhosis and HPS than in those without HPS (8), and that AaPO2 was linearly related to exhaled NO concentrations (1). In addition, the intrapulmonary vascular dilatations and increases in both exhaled NO concentrations and AaPO2 resolved after liver transplantation (9). These findings suggest that NO overproduction in the lungs may be the mechanism underlying HPS and the associated pulmonary vasoreactivity blunting.
The aim of the present study was to determine whether the common-bile-duct-ligation rat model is associated with pulmonary NO overproduction replicating that seen in patients with HPS. Assuming such an increase was found, additional goals were to investigate which nitric oxide synthase (NOS) isoform was activated in the lungs and to determine the role of NO overproduction in cirrhosis-related HPS. Thus, we measured exhaled NO concentrations and NOS activities and expression in the lungs of rats with cirrhosis. We also investigated whether chronic inhibition of lung NOS activity with the nonspecific inhibitor NG-nitro-L-arginine methyl ester (L-NAME) prevented HPS in cirrhotic rats.
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Animals
Male Wistar rats weighing 200 to 300 g were used in all experiments.
Common bile duct ligation was performed as previously described
(10). The rats were divided into four groups: an untreated control sham
group, a control sham group treated with L-NAME (control-L-NAME), an untreated cirrhotic group, and a cirrhotic group treated
with L-NAME (cirrhotic-L-NAME). L-NAME was given orally for 6 wk at a dose of 5 mg · kg1 · d1. All animals were studied 6 wk after
common bile duct ligation or sham operation.
Hemodynamic and Blood Gas Measurements
Two days before the hemodynamic study, the rats were anesthetized
using intraperitoneal ketamine (100 mg · kg1) and xylazine (0.75 mg · kg1). An umbilical vessel catheter was inserted and moved through the right ventricle into the pulmonary artery. A thermistor was positioned in the aortic arch and another polyethylene catheter was inserted into the tail artery for systemic arterial pressure measurements
and arterial blood gas analysis. Cardiac output was measured using a
thermodilution method. AaPO2 was calculated using the modified alveolar gas equation. Baseline hemodynamic and blood gas parameters were recorded 30 min after each animal was placed in a study
chamber continuously flushed with compressed air. The study chamber was then flushed with a hypoxic gas mixture (10% O2, 90% N2)
for 30 min. Thirty minutes after the hypoxic challenge, angiotensin II
was infused in a dose of 100 mg · kg1 · min1 for 8 min.
Detection of Intrapulmonary Vascular Dilatations
Two hundred µCi of 99mTc-labeled albumin macroaggregates (mean size = 20 µm, range 15 to 50 µm) were injected through a jugular catheter. Thirty minutes after the injection, lung and brain radioactivities were measured (11) using a gamma detector, and ratio of brain-over-lung radioactivity was calculated.
NO Measurements
Rats were ventilated with room air through a tracheotomy. Exhaled air was collected in a 3-L polyethylene bag for 15 min. The NO concentration was analyzed using a chemoluminescence NO analyzer (NOX 4000 EVA; Seres, Aix-en-Provence, France). Four untreated and five L-NAME-treated cirrhotic rats underwent exhaled NO measurement and isotopic investigation simultaneously.
Lung NOS Activity Measurements
Total and calcium-independent NOS activities were measured by determining the conversion of [14C]L-arginine to [14C]L-citrulline as previously reported (12).
Lung NOS Protein Expression
Lung endothelial NOS (eNOS) and inducible NOS (iNOS) protein expressions were determined with specific antisera against eNOS and iNOS (Transduction Laboratories, Lexington, U.K.). Densitometry results are expressed as a percentage of the value in untreated controls (100%).
Microscopic Examination
Distended lungs were fixed by intratracheal infusion of 10% formalin at a pressure of 25 cm H2O and tissue samples were counterstained with hematoxylin.
Immunohistochemistry
Immunolocalization of macrophages was performed using the anti-rat monoclonal macrophages antibody ED1 (Biosource International, Camarillo, CA) on paraffin sections. Immunolocalization of iNOS and eNOS was performed on frozen sections using monoclonal antibodies (Transduction Laboratories, Lexington, U.K.).
Statistical Analysis
Results were analyzed using linear correlation and analysis of variance followed by Student-Newman-Keuls post hoc tests. All values are expressed as mean ± SEM. Values of p < 0.05 were considered statistically significant.
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RESULTS |
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The untreated and L-NAME-treated cirrhotic rats became jaundiced, and most had ascites and a micronodular liver. The diagnosis of cirrhosis was confirmed based on macroscopic features and histologic findings at autopsy. Less than 1% of rats died after surgery. The 5 wk mortality rates were similar in untreated and L-NAME-treated cirrhotic rats (25% versus 22%, respectively).
All lungs from untreated and L-NAME-treated cirrhotic rats showed accumulation of large mononuclear macrophage-like cells within the lumen of numerous small muscular and nonmuscular pulmonary vessels (Figure 1). The lumen of some vessels appeared nearly obliterated by cell clusters. These cells were strongly immunoreactive for the specific rat macrophage monoclonal antibody ED1 (Figure 1). They were not present in the untreated or L-NAME-treated control rats.
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Pulmonary Hemodynamics and Pulmonary Vascular Reactivity in Cirrhotic Rats
Untreated cirrhotic rats exhibited a characteristic hemodynamic pattern of pulmonary and systemic arterial vasodilatation with hyperdynamic circulation, as indicated by lower pulmonary and systemic vascular resistance values and higher cardiac index values compared with the other groups (Table 1). L-NAME-treated cirrhotic rats had higher total pulmonary and systemic vascular resistance values and lower cardiac index values than untreated cirrhotic rats (Table 1). The hemodynamic values in L-NAME-treated cirrhotic rats were similar to those in untreated control rats (Table 1). L-NAME-treated control rats had higher pulmonary artery pressure and pulmonary vascular resistance values than the three other groups.
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Untreated cirrhotic rats had a noticeably depressed pressor response to hypoxia (10% fraction of inspired oxygen compared with untreated control rats (Table 2). Chronic L-NAME administration restored a normal hypoxic pressor response in cirrhotic rats (Table 2). The pressor response to angiotensin II was also reduced in untreated cirrhotic rats and normalized by chronic L-NAME administration, as compared with untreated control animals (Table 2). L-NAME-treated control animals exhibited a stronger pressor response to hypoxia and angiotensin II than the three other groups.
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HPS in Cirrhotic Rats
Assessment of HPS in cirrhotic rats was based on the combination of gas exchange abnormalities and intrapulmonary vascular dilatations. Untreated cirrhotic rats had lower PaO2 and higher AaPO2 values than untreated control rats (Table 1). PaCO2 was similar in these two groups (Table 1). The ratio of brain-over-lung radioactivities increased markedly in all untreated cirrhotic rats, reflecting the presence of intrapulmonary vascular dilatations (Figure 2).
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L-NAME normalized PaO2 and AaPO2 levels (Table 1), as well as the brain-over-lung radioactivity ratio (Figure 2), indicating that chronic L-NAME administration prevented HPS in cirrhotic rats. L-NAME did not change significantly the brain-over-lung radioactivity ratio in control rats.
Lung NO Production
Compared with untreated control rats, untreated cirrhotic rats showed a 2.7-fold increase in exhaled NO concentrations (Figure 3). L-NAME treatment returned exhaled NO concentrations to the levels seen in untreated control animals. Although not significant, a decrease in exhaled NO concentrations was observed in L-NAME-treated control rats. When the brain-over-lung radioactivity ratio and exhaled NO concentration data from untreated and L-NAME-treated cirrhotic rats were pooled in a simple linear regression analysis, a significant correlation was found (r = 0.79, p = 0.012): the higher the NO concentration, the higher the ratio of brain-over-lung radioactivity.
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Total and calcium-independent NOS activities in the lungs showed a 1.7-fold and a 5.3-fold increase, respectively, in untreated cirrhotic compared with untreated control rats (Figure 4). Total and calcium-independent lung NOS activities were correlated with each other in untreated cirrhotic rats (Figure 5, y = 1.7x + 3.8, r = 0.98, p = 0.0005). Total lung NOS activity was normal in L-NAME-treated cirrhotic rats, but calcium-independent NOS activity remained slightly elevated as compared with untreated control rats (Figure 4). L-NAME-treated control rats had lower total lung NOS activities than the three other groups (data not shown).
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Significantly different from untreated
controls (p < 0.05). 
Significantly different from untreated controls (p < 0.05).
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Lung NOS Expression
Levels of eNOS protein expression in the lungs increased in untreated cirrhotic rats (189 ± 20% of untreated control values, p < 0.01) and rose further with chronic L-NAME treatment (288 ± 31% of untreated control values, p < 0.01) (Figure 6). Similarly, eNOS expression increased 1.6-fold in L-NAME-treated control rats.
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Levels of iNOS protein expression showed an increase in the lungs of untreated cirrhotic rats (161 ± 10% of untreated control values, p < 0.01) and an even greater elevation with chronic L-NAME treatment (284 ± 30% of untreated control values, p < 0.01) (Figure 7). Levels of iNOS expression were unaffected by chronic L-NAME treatment in control rats.
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Immunostaining of the lungs with antibody specific for eNOS showed similar positive immunoreactivity in pulmonary endothelial cells in untreated control rats, L-NAME-treated control rats, untreated cirrhotic, and L-NAME-treated cirrhotic rats.
Immunostaining of the lungs of untreated cirrhotic rats and L-NAME-treated cirrhotic rats with antibody specific for iNOS showed marked positive immunoreactivity in intravascular macrophages (Figure 1). By contrast, only sparse signals were found after immunostaining of the lungs of untreated and L-NAME-treated control animals with antibody specific for iNOS.
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DISCUSSION |
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The goals of the present research study on the role of NO in the pathogenesis of HPS was to identify the specific NOS isoform (i.e., constitutive or inducible NOS) responsible for NO overproduction in the lungs, to clarify the role of NO in the pathogenesis of intrapulmonary vascular dilatations and gas exchange alterations, and to determine the mechanisms of NOS activation. The results showed that HPS development in untreated cirrhotic rats, defined as the presence of intrapulmonary vascular dilatations with increased AaPO2 values, was associated with NO overproduction in the lungs due to accumulation of intravascular macrophages expressing iNOS and, to a lesser extent, to increased eNOS expression. Normalization of NO lung production with chronic L-NAME administration prevented the development of HPS and corrected both the blunting of the pressor response to pulmonary vasoconstrictors and the hyperdynamic circulation. Thus, iNOS and eNOS overexpression in the lungs led to increased local NO production, which contributed to the genesis of HPS with the attendant pulmonary hemodynamic abnormalities.
In this study, we used a well-characterized animal model of cirrhosis resulting in HPS (4, 5, 15). Intrapulmonary vascular dilatations were detected in vivo using the 99mTc-labeled albumin macroaggregate technique, a well-established method (11) for identifying intrapulmonary vascular dilatations in patients with hypoxemia and liver disease. Brain radionuclide uptake was substantially increased as compared with untreated controls in all untreated cirrhotic rats (Figure 2), indicating that pulmonary capillaries, normally the narrowest segment of the pulmonary circulation, were broadened or bypassed, allowing passage of albumin macroaggregates larger than 15 µm in diameter. This confirms a study conducted by Fallon and coworkers using a similar technique in cirrhotic rats (5). Schraufnagel and coworkers found an increase in alveolar capillary diameter in cirrhotic rats that was too small (< 1 µm) to allow passage of macroaggregates (16). Alternatively, passage of macroaggregate may occur through pulmonary artery to pulmonary vein anastomoses (17, 18). Hypoxemia and intrapulmonary vascular dilatations were found in all untreated cirrhotic rats, suggesting that hypoxemia was at least partly related to intrapulmonary shunting. Furthermore, these rats with HPS had hyperdynamic circulation, arterial vasodilatation, and decreased pulmonary vascular tone. The decrease in pulmonary vascular tone was indicated by lower levels of baseline pulmonary vascular resistance, blunted pulmonary vasoconstriction in response to hypoxia, and reduced sensitivity to the pulmonary vasoconstrictor angiotensin II, as in an earlier study by Chang and Ohara (4).
A role for NO in the pulmonary vasodilation seen in cirrhosis has been suggested by previous studies (15, 19) showing enhanced basal NO activity and NO-mediated impairment in the vasoconstrictive response to phenylephrine in intralobar pulmonary artery rings from cirrhotic rats. Similar findings have been reported in the systemic circulation (20). However, no studies have measured pulmonary NO production or its effect on gas exchange, development of intrapulmonary vascular dilatations, and pulmonary hemodynamics in cirrhotic rats. In the present study, exhaled NO concentrations were markedly increased in all untreated cirrhotic rats, with a mean value of 11.1 ± 1.6 parts per billion (ppb), which is similar to that reported in patients with cirrhosis (9). This increase in exhaled NO was associated with increases in total and calcium-independent NOS activities in lung homogenates. Total NOS activity exhibited a strong linear correlation with calcium-independent NOS activity in the lungs of untreated cirrhotic rats, suggesting that overexpression of iNOS contributed most of the increase in total NOS activity.
Controversy exists about which NOS isoforms are involved in increased lung NO production in liver disease. Our group and others have shown that expression of eNOS, but not iNOS, is increased in the systemic vascular bed (20, 21), explaining the greater endothelium-dependent relaxation in systemic arteries in untreated cirrhotic rats than in untreated control rats (22). In contrast, we (19) and others (15) have demonstrated that pulmonary artery endothelium-dependent relaxations are similar in untreated cirrhotic and control rats, suggesting that iNOS rather than eNOS overexpression causes the NO activity increase seen in pulmonary arteries after the development of cirrhosis. In two studies using immunohistochemistry and Northern blot analysis to detect iNOS and eNOS in cirrhotic rat lungs, Mizumoto and coworkers (23) demonstrated an increase in the expression of iNOS, but not in eNOS, after the induction of cirrhosis by carbon tetrachloride, whereas Fallon and coworkers (15) found the opposite after biliary cirrhosis. In keeping with our NOS activity results, we found that iNOS and, to a lesser extent, eNOS were increased in the lungs of animals with cirrhosis. Immunohistochemistry demonstrated increased expression of iNOS within intravascular macrophages. Thus, our data are consistent with increased expression and activity of iNOS and, to a lesser extent, eNOS in the lungs of untreated cirrhotic rats, with increased pulmonary NO production as a result. We did not measure neuronal NOS expression in this study because there is evidence that pulmonary vascular tone in rodents is modulated by eNOS-derived and iNOS-derived NO, but not neuronal NOS-derived NO (24).
To investigate the effects on HPS in cirrhotic rats of normalizing pulmonary NO production, the nonspecific NOS inhibitor L-NAME was administered orally for 6 wk after common bile duct ligation. Because progressive intrapulmonary
vascular dilatations begin to develop within 2 wk after common bile duct ligation in rats (5), L-NAME administration was
started immediately after common bile duct ligation in our
study. The L-NAME dose was 5 mg · kg1 · d1. This dose was
chosen based on previous evidence that an L-NAME dose of 3 mg · kg1 · d1 reduced the cardiac index and aortic cyclic guanosine monophosphate concentrations to near-normal levels
in cirrhotic rats (25). We believe that this dosage was adequate
because L-NAME normalized lung NO production, decreasing both exhaled NO concentrations and total NOS activity to
levels similar to those in untreated control rats.
Calcium-independent NOS activity remained slightly elevated after L-NAME, suggesting persistent iNOS expression. Interestingly, L-NAME increased lung iNOS and eNOS proteins to levels above those in untreated cirrhotic rats. An increase in iNOS protein expression after administration of NOS inhibitors has been previously demonstrated in liver (26), glial cells (27), or endocrine pancreas (28). In addition, several studies have shown that expressions of constitutive and inducible NOS (29, 30) are decreased by exogenous NO. All these results are in keeping with our findings and indicate that iNOS and eNOS protein expression may be subject to end-product feedback regulation by NO. Chronic L-NAME administration corrected systemic vasodilatation and hyperdynamic circulation in our rats with common bile duct ligation-induced cirrhosis, as previously demonstrated in rats with carbon tetrachloride-induced cirrhosis (25). As expected, L-NAME also normalized pulmonary vascular resistance and restored pulmonary vasoconstrictor responses to hypoxia and angiotensin II. Thus contrary to the control rats treated with L-NAME that developed systemic and pulmonary hypertension, there were no detrimental hemodynamic side effects with L-NAME in cirrhotic rats.
These findings extend to in vivo conditions the previous observation that L-NAME normalized the depressed contractile response of pulmonary artery rings from cirrhotic rats (15, 19). Our results, however, differ from that of Carter and coworkers (31) who reported that acute administration of NOS inhibitor failed to restore normal hypoxic pulmonary vasoconstriction in isolated lungs of cirrhotic rats. This indicates that a chronic L-NAME treatment rather than an acute administration of NOS inhibitor is required to normalize NO production and restore the blunted pulmonary vasoreactivity in cirrhotic rats. Thus, NO overproduction in cirrhotic rats results in both pulmonary and systemic vasodilatation. Prevention of the development of intrapulmonary vascular dilatations, as assessed by the brain-over-lung radioactivity ratio, was observed in all cirrhotic rats treated with L-NAME, suggesting that the extent of intrapulmonary vascular dilatations was related to the level of pulmonary NO production. The positive linear correlation between the brain-over-lung radioactivity ratio and exhaled NO concentrations in untreated cirrhotic rats supports this hypothesis.
The mechanisms by which NO contributes to the development of capillary dilatations and pulmonary-systemic microvascular connections in cirrhotic rats need to be clarified. Pulmonary capillary dilatation or the opening of precapillary vascular communications may occur as a result of distention or recruitment related to increased pulmonary blood flow (32). In addition, NO may directly enhance pulmonary microvascular angiogenesis independently from its vasodilating action (33). NO has been shown to exert both proliferative and antiproliferative effects on pulmonary vascular cells. In chronic hypoxia exogenous NO given by inhalation reduces pulmonary vascular remodeling (36), whereas endogenous NO is critical for the proliferation of pulmonary vascular smooth muscle cells (37). Thus, site of origin of NO and its concentration are factors that may influence which action NO shows, and endogenous NO derived from eNOS and iNOS is generally considered as a mediator of pulmonary angiogenesis. These observations explained why chronic inhaled NO, that is given at supra-physiologic concentrations, does not reproduce by itself the features of HPS. Prevention of intrapulmonary vascular dilatations with L-NAME was consistently associated with gas exchange normalization in our study. These results agree with reports of correlation between AaPO2 and exhaled NO in patients with cirrhosis (8) and between the extent of AaPO2 reductions and exhaled NO after liver transplantation (9).
Factors other than direct pulmonary NO inhibition may be implicated also. Chronic L-NAME administration may influence the development of portal hypertension and the natural history of obstructive hepatic injury, thus altering the hepatic production and metabolism of other mediators. However, we and others have shown than NOS inhibition significantly affects systemic hemodynamics without influencing portal circulatory abnormalities (38, 39). Moreover, although we did not specifically evaluate the severity of hepatic injury, all L-NAME-treated rats with common bile duct ligation became jaundiced, most had ascites, and all had histologic evidence of biliary cirrhosis; furthermore, the mortality rate was similar in the untreated and L-NAME-treated cirrhotic rats.
In contrast to our finding of iNOS overexpression in the lungs of untreated cirrhotic rats, our group and others failed to detect iNOS expression in the peripheral systemic vascular bed of cirrhotic rats (20, 21). We speculate that increased iNOS expression and, to a lesser extent, eNOS may be the result of increased exposure of cirrhotic rat lungs to endotoxins or cytokines (40, 41). Bile duct ligation in rats results in bacterial translocation from the gastrointestinal tract and in endotoxemia, a phenomenon that exposes the lungs to the effects of endotoxins and proinflammatory cytokines (42, 43). A recent study by Chang and Ohara (44) showed a fivefold increase in lung uptake of circulating endotoxins in cirrhotic rats, with almost no change in endotoxin uptake in the blood, liver, spleen, or kidneys. This increase in pulmonary circulating endotoxin uptake was ascribable to marked accumulation of intravascular macrophages in the pulmonary microcirculation of cirrhotic rats in studies by Schraufnagel and Kay (17) and Chang and Ohara (44). Extensive accumulation of pulmonary intravascular macrophages was observed consistently in our untreated cirrhotic rats (Figure 1), and iNOS was expressed in these pulmonary intravascular macrophages (Figure 1), as recently shown in circulating monocytes-macrophage cells in cirrhotic patients (45). We hypothesize that high concentrations of endotoxins or cytokines (or both) in the pulmonary bloodstream activate pulmonary intravascular macrophage recruitment and iNOS expression, thus increasing the amount of NO released in the lungs of subjects with cirrhosis.
Our study has important implications. First, normalization of NOS activity with L-NAME corrected hyperdynamic circulation and prevented HPS, suggesting that increased NOS activity may play an important role in systemic and pulmonary vasodilatation, rather than being a response to the increased shear stress caused by hyperdynamic circulation. Second, modulation of NO production, which was recently suggested as an approach to HPS management (46), may be harmful because withdrawal of the NOS inhibitor may be followed by NOS protein overexpression and NO overproduction. Third, the preferential expression of iNOS in the lungs of untreated cirrhotic rats suggests that the lung may be a preferred target for endotoxin or cytokines in cirrhosis.
In conclusion, development of HPS in untreated cirrhotic rats was associated with increased pulmonary NO production resulting from increased expression and activity of intravascular macrophage iNOS and, to a lesser extent, lung eNOS. Normalization of NO lung production with chronic L-NAME treatment prevented HPS and corrected the hyperdynamic circulation and blunted response to pulmonary vasoconstrictors, but was associated with overexpression of iNOS and, to a lesser degree, eNOS. Our findings strongly suggest that increased pulmonary NO production plays a role in the pathogenesis of HPS and of the associated pulmonary hemodynamic abnormalities in cirrhotic rats.
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Footnotes |
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Correspondence and requests for reprints should be addressed to P. Hervé, M.D., Centre Chirurgical Marie Lannelongue, 133, avenue de la Résistance, 92350 Le Plessis Robinson, France. E-mail: pherve{at}ccml.com
(Received in original form September 8, 2000 and in revised form January 30, 2001).
H. Nunes held a fellowship from the Fondation pour la Recherche Médicale and J. Heller from the Fondation Nationale Alfred Kastler and Ernst und Berta Grimmke-Stiftung.
Acknowledgments:
The authors wish to thank Odile Poirel for her excellent
technical assistance.
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References |
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REFERENCES
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