Impact of liver damage on blood-borne variables and pulmonary hemodynamic responses to hypoxia and hyperoxia in anesthetized rats.

BACKGROUND: Liver disorders may be associated with normal pulmonary hemodynamic, hepatopulmonary syndrome (HPS), or portopulmonary hypertension (POPH). In this study, we aimed to investigate the effect of the severity of liver dysfunctions on blood-borne variables, and pulmonary hemodynamic during repeated ventilation with hyperoxic and hypoxic gases. METHODS: Female Sprague Dawley rats were assigned into four groups of Sham (n = 7), portal vein ligation (PPVL, n = 7), common bile duct ligation (CBDL, n = 7), and combination of them (CBDL+ PPVL, n = 7). Twenty-eight days later, right ventricular systolic pressure (RVSP) and systemic blood pressure were recorded in anesthetized animals subjected to repeated maneuvers of hyperoxia (O2 50%) and hypoxia (O2 10%). Besides, we assessed blood parameters and liver histology. RESULTS: Liver histology score, liver enzymes, WBC and plasma malondialdehyde in the CBDL+PPVL group were higher than those in the CBDL group. Also, the plasma platelet level in the CBDL+PPVL group was lower than those in the other groups. On the other hand, the serum estradiol in the CBDL group was higher than that in the CBDL+PPVL group. All the above parameters in the PPVL group were similar to those in the Sham group. During ventilation with hyperoxia gas, RVSP in the CBDL+PPVL group was higher than the ones in the other groups, and in the CBDL group, it was more than those in the PPVL and Sham groups. Hypoxic pulmonary vasoconstriction (HPV) was not detected in both CBDL+PPVL and CBDL groups, whereas, it retained in the PPVL group. CONCLUSION: Severe liver damage increases RVSP in the CBDL+PPVL group linked to the high level of ROS, low levels of serum estradiol and platelets or a combination of them. Furthermore, the high RVSP at the noted group could present a reliable animal model for POPH in female rats.

Loss of cell polarity regulated by PTEN/Cdc42 enrolled in the process of Hepatopulmonary Syndrome.

One central factor in hepatopulmonary syndrome (HPS) pathogenesis is pulmonary vascular remodelling (PVR) which involves dysregulation of proliferation and migration in pulmonary microvascular endothelial cells (PMVECs). Growing evidence suggests that Apical/basolateral polarity plays an important role in cell proliferation, migration, adhesion and differentiation. In this study, we explored whether cell polarity is involved and critical in experimental HPS rats that are induced by common bile duct ligation (CBDL). Cell polarity related proteins were analysed in CBDL rats lung and PMVECs under the HPS serum stimulation by immunofluorescence assay. Cdc42/PTEN activity, cell proliferation and migration and Annexin A2 (AX2) in PMVECs were determined, respectively. Cell polarity related proteins, lost their specialized luminal localization in PMVECs of the CBDL rat. The loss of cell polarity was induced by abnormal activity of Cdc42, which was strongly enhanced by the interaction between p-PTEN and Annexin A2 in PMVECs, after treatment with serum from CBDL rats. It led to over-proliferation and high migration ability of PMVECs. Down-regulation of PTEN-Cdc42 activity in PMVECs restored cell polarity and thus reduced their ability of migration and proliferation. Our study suggested that the loss of cell polarity plays a critical role in the pathogenesis of HPS-associated PVR and may become a potentially effective therapeutic target.

Sorafenib in Hepatopulmonary Syndrome: A Randomized, Double-Blind, Placebo-Controlled Trial.

The tyrosine kinase inhibitor sorafenib improves hepatopulmonary syndrome (HPS) in an experimental model. However, the efficacy and adverse effect profile in patients with HPS are unknown. We aimed to determine the effect of sorafenib on the alveolar-arterial oxygen gradient (AaPO2 ) at 3 months in patients with HPS. We performed a randomized, double-blind, placebo-controlled parallel trial of sorafenib in patients with HPS at 7 centers. A total of 28 patients with HPS were randomized to sorafenib 400 mg by mouth daily or a matching placebo in a 1:1 ratio. We found no statistically significant difference in the median change in AaPO2 from baseline to 12 weeks between the patients allocated to sorafenib (4.5 mm Hg; IQR, -3.8 to 7.0 mm Hg) and those allocated to placebo (-2.4 mm Hg; IQR, -4.8 to 8.2 mm Hg; P = 0.70). There was also no difference between the groups in terms of degree of intrapulmonary shunting by contrast echocardiography. Sorafenib significantly reduced circulating levels of angiogenic markers, including vascular endothelial growth factor receptors (P < 0.01) and TIE2-expressing M2 monocytes (P = 0.03), but it reduced the mental component scores of the Short Form 36 (P = 0.04), indicating a worse quality of life. In conclusion, sorafenib did not change the AaPO2 or other disease markers at 3 months in patients with HPS. Alternative antiangiogenic therapies or treatments targeting other pathways should be investigated.

Does Biliodigestive Anastomosis Have Any Effect on the Reversal of Hepatopulmonary Syndrome in a Biliary Cirrhosis Experimental Model?

BACKGROUND: Biliary cirrhosis is associated with hepatopulmonary syndrome (HPS), which is related to increased posttransplant morbidity and mortality. AIMS: This study aims to analyze the pathophysiology of biliary cirrhosis and the onset of HPS. METHODS: Twenty-one-day-old Wistar rats were subjected to common bile duct ligation and were allocated to two groups: group A (killed 2, 3, 4, 5, or 6 weeks after biliary obstruction) and group B (subjected to biliodigestive anastomosis 2, 3, 4, 5, or 6 weeks after the first procedure and killed 3 weeks later). At the killing, arterial blood was collected for the analyses, and samples from the liver and lungs were collected for histologic and molecular analyses. The gasometric parameters as well as the expression levels of ET-1, eNOS, and NOS genes in the lung tissue were evaluated. RESULTS: From a total of 42 blood samples, 15 showed hypoxemia (pO2 < 85 mmHg) and 17 showed an increased oxygen gradient [p (A-a) O2 > 18 mmHg]. The liver histology revealed increased ductular proliferation after common bile duct ligation, and reconstruction of bile flow promoted decreased ductular proliferation 5 and 6 weeks post-common bile duct ligation. Pulmonary alterations consisted of decreased parenchymal airspace and increased medial wall thickness. Biliary desobstruction promoted transitory improvements 5 weeks after biliary obstruction (increased parenchymal airspace and decreased MWT-p = 0.003 and p = 0.004, respectively) as well as increased endothelin expression levels (p = 0.009). CONCLUSIONS: The present model showed lung tissue alterations promoted by biliary obstruction. The biliodigestive anastomosis had no clear direct effects on these alterations.

Annexin A2-modulated proliferation of pulmonary arterial smooth muscle cells depends on caveolae and caveolin-1 in hepatopulmonary syndrome.

We have established that annexin A2 (ANXA2) is an important factor in the experimental hepatopulmonary syndrome (HPS) serum-induced proliferation of pulmonary arterial smooth muscle cells (PASMCs). However, the detailed mechanism remains unclear. ANXA2 translocated to the caveolin-enriched microdomains (caveolae) in PASMCs upon HPS serum stimulation. The disruption of caveolae by Methyl-ß-cyclodextrin (MßCD) alleviated the caveolae recruitment of ANXA2 and the ANXA2-mediated activation of ERK1/2 and NF-κB, so that ANXA2-modulated PASMC proliferation was suppressed. The over-expression of Cav-1 resulted in the relocation of ANXA2 from caveolae and negatively regulated ERK1/2 and NF-κB activation, which inhibited the ANXA2-modulated PASMC proliferative behavior. These data indicate that caveolae function as a signaling platform for ANXA2-induced proliferative behavior and Cav-1 participates upstream of ANXA2 in the activation of ERK1/2 and NF-κB.

Isolated Intrapulmonary Vascular Dilatations and the Risk of Developing Hepatopulmonary Syndrome in Liver Transplant Candidates.

BACKGROUND: The natural history of intrapulmonary vascular dilations (IPVD) and their impact on patient outcomes in the setting of portal hypertension has only been described in small series. AIMS: To assess the development of hepatopulmonary síndrome (HPS) in patients with isolated IPVD and to evaluate outcomes of IPVD and HPS among patients evaluated for liver transplantation (LT). MATERIAL AND METHODS: Data from a prospective cohort of patients evaluated for LT with standardized screening for HPS were analyzed. IPVDs were defined as the presence of microbubbles in the left atrium > 3 cycles following right atrial opacification. HPS was defined as the presence of IPVD and hypoxemia (Alveolar-arterial gradient ≥ 15 mmHg) in the absence of concomitant cardiopulmonary disease. RESULTS: A total of 104 patients with negative contrast-enhanced echocardiogram (CE) were compared to 63 patients with IPVD and 63 patients with HPS. Only four patients were categorized as 'severe' HPS based on degree of hipoxemia (defined as PaO2 < 60 mmHg). Twenty IPVD patients were followed with ABG over a mean duration of 21 months (range 9-43), of whom 7 (35%) subsequently met HPS criteria. Overall unadjusted survival from the time of LT evaluation using multi-state survival models that accounted for pre- and post-LT time was not statistically different among the three groups (negative CE, IPVD, and HPS; p > 0.5). CONCLUSIONS: Patients with IPVD appear to have a substantial risk of developing oxygenation impairment over time and progress to HPS. In our cohort, survival in patients with HPS and isolated IPVD is not different when compared to those without IPVDs.

Serum bile acids in patients with hepatopulmonary syndrome.

Background Hepatopulmonary syndrome (HPS) occurs in 20 - 30 % of patients with cirrhosis and is associated with increased mortality. Cholestasis and accumulation of bile acids (BAs) play a major role in chronic liver disease. Aim We aimed to evaluate the clinical role of serum BAs in patients with HPS. Methods Seventy-four patients with cirrhosis were included in this prospective study. Marker for cholestasis as total and individual serum BAs, bilirubin, alkaline phosphatase (AP), and gamma-glutamyl transpeptidase (GGT) were analyzed in patients screened for HPS. Criteria of HPS were fulfilled in 26 patients (35 %). Results In contrast to AP and GGT, bilirubin and serum BAs were significantly elevated in patients with HPS (median total BAs in HPS 83.5 µmol/L, IQR 43.1 - 148.9 vs. no HPS 26.9 µmol/L, 11 - 75.6; p < 0.001). Total BAs correlated with gas exchange by means of PaO2 / AaPO2 (r: -0.28, p < 0.05; r: 0.24, p < 0.05) and portal pressure (r: 0.33, p < 0.05). BAs were associated with HPS independently severity of underlying liver disease (OR: 1.012, 95 % CI: 1.001 - 1.023, p < 0.05). Conclusion BA retention is associated with HPS and gas exchange abnormalities. Future studies should assess whether modulation of BAs signaling may impact the course of HPS.

Hepatopulmonary syndrome: which blood gas analysis criteria and position should we use for diagnosis?

INTRODUCTION: Different blood gas criteria have been used in the diagnosis of hepatopulmonary syndrome (HPS). PATIENTS AND METHODS: Arterial blood gases were prospectively evaluated in 194 cirrhotic candidates for liver transplantation (LT) in the supine and seated position. Three blood gas criteria were analyzed: classic (partial pressure of oxygen [PaO2] < 70 mmHg and/or alveolar-arterial gradient of oxygen [A-a PO2] ≥ 20 mmHg), modern (A-a PO2 ≥ 15 mmHg or ≥ 20 mmHg in patients over 64) and the A-a PO2 ≥ threshold value adjusted for age. RESULTS: The prevalence of HPS in the supine and seated position was 27.8% and 23.2% (classic), 34% and 25.3% (modern) and 22.2% and 19% (adjusted for age), respectively. The proportion of severe and very severe cases increased in a seated position (11/49 [22.4%] vs 5/66 [7.6%], p = 0.02). No difference was observed in the pre-LT, post-LT and overall mortality in patients with HPS, regardless of the criteria used. CONCLUSION: Obtaining blood gas measurements in the supine position and the use of modern criteria are more sensitive for the diagnosis of HPS. Blood gas analysis with the patient seated detects a greater number of severe and very severe cases. The presence of HPS was not associated with an increase in mortality regardless of blood gas criterion used.

Nitric oxide metabolites, nitrative stress, and paraoxonase activity in hepatopulmonary syndrome.

AIM: To investigate possible abnormalities of vasoactive compounds, nitrative stress, and antioxidant activity of paraoxonase (PONa) in human hepatopulmonary syndrome (HPS), we determined endothelin-1 (ET), nitric oxide (NOx) metabolites, PONa alongside crude plasma nitrotyrosine (NT) as surrogate marker of nitrative stress. MATERIAL AND METHODS: Liver cirrhosis (LC) patients with HPS (n = 12) were matched by age, sex, and Child-Pugh score to LC patients without HPS (n = 15) and to healthy controls (CTR) (n = 15); plasma NO2(-) (nitrite) (vascular metabolite), NO3(-) (nitrate) (inflammatory metabolite), and PONa were determined by a colorimetric assay, ET, and NT by immunoassays. RESULTS: HPS patients showed higher level of ET (p = 0.0002), NO2(-) (p = 0.002), NO3(-) (p = 0.0001), NT (p < 0.0001), and lower PONa (p = 0.0004) than CTR; post-hoc analysis revealed greater ET (p < 0.05) and NO3(-) (p < 0.005) in LC patients with HPS than in LC patients without HPS. NT correlated to Child-Pugh score within HPS (p = 0.04) and LC (p = 0.02). CONCLUSION: Our HPS patients are characterized by elevated plasma levels of ET and NOx metabolites and lower PONa. Reduced PONa alongside elevated NO3(-) and NT suggests that defective antioxidation may favor nitrative stress and both may be implicated in the pathogenesis of HPS.

Role of angiogenic factors/cell adhesion markers in serum of cirrhotic patients with hepatopulmonary syndrome.

BACKGROUND & AIMS: Hepatopulmonary syndrome is a complication of chronic liver disease resulting in increased morbidity and mortality. It is caused by intrapulmonary vascular dilations and arteriovenous connections with devastating influence on gas exchange. The pathogenesis is not completely understood but evidence mounts for angiogenesis. Aims of this study were to identify angiogenic factors in serum of patients with hepatopulmonary syndrome and to study the possibility to predict its presence by these factors. METHODS: Multiplex assays were used to measure the concentration of angiogenic factors in patients with (n = 30) and without hepatopulmonary syndrome (n = 30). Diagnosis was based on the presence of gas exchange abnormality and intrapulmonary vasodilations according to published guidelines. RESULTS: Patients with and without hepatopulmonary syndrome had similar MELD scores (median: 11.2 vs. 11.6; P = 0.7), Child-Pugh score (P = 0.7) and PaCO2 values (median: 35 vs. 37; P = 0.06). PaO2 and P(A-a) O2 gradient were significantly different (respectively median of 80 vs. 86, P = 0.02; and 24 vs. 16, P = 0.004). Based on area under the curve (AUC) data and P-values, the best predictors were vascular cell adhesion molecule 1 (VCAM1) (AUC = 0.932; P < 0.001) and intercellular adhesion molecule 3 (ICAM3) (AUC = 0.741; P = 0.003). Combining these factors results in an AUC of 0.99 (after cross-validation still 0.99). CONCLUSIONS: VCAM1 and ICAM3 might be promising biomarkers for predicting hepatopulmonary syndrome. Combining these factors results in an AUC of 0.99 and a negative predictive value of 100%. Determining the concentration of these biomarkers might be a screening method to detect hepatopulmonary syndrome. The use of these biomarkers should be validated in larger groups of patients.

Variability of oxygenation in possible hepatopulmonary syndrome: effects of requiring two abnormal arterial blood gas results for diagnosis.

BACKGROUND AND AIMS: Hepatopulmonary syndrome (HPS) affects 10-32 % of patients with cirrhosis and is defined by liver abnormalities, intrapulmonary vascular dilatations (IPVDs), and abnormal oxygenation. However, published criteria for abnormal oxygenation are inconsistent. We sought to evaluate variation in oxygenation over time and to compare various diagnostic criteria for validity, based on their diagnostic stability over time and ability to identify patients with clinically relevant findings. METHODS: We retrospectively analyzed oxygenation and diffusion capacity in patients with liver abnormalities and IPVDs who had ≥ 2 arterial blood gases (ABGs) at the University of Toronto or Universite de Montreal. We compared the performance of nine possible oxygenation criteria for HPS and for each explored whether validity improved when requiring two consecutive abnormal ABGs on different days. RESULTS: Mean PaO2 was 68.4 mmHg and annual within-patient coefficient of variation 6.3 % (58 patients). Applying published criteria, 8.6-15.5 % of patients initially diagnosed with HPS no longer met the criterion for HPS on a subsequent ABG (re-classified). Requiring two consecutive abnormal ABGs on different days: (1) reduced the proportion of re-classified patients (9/9 criteria); (2) identified patients with more rapid progression in hypoxemia and greater difference in rate of progression between HPS and non-HPS (7/9 criteria); and (3) identified patients with lower diffusion and a larger difference in diffusion between HPS and non-HPS (8/9 criteria). CONCLUSIONS: Oxygenation is variable in this population, and requiring two abnormal results might reduce misdiagnosis and better differentiate patients with and without HPS according to clinically relevant markers of disease.

[Hepatopulmonary syndrome-related changes in D-dimer, prothrombin time, fibrinogen, CD4 and CD8 in a rat model system].

OBJECTIVE: To determine the changes in levels of D-dimer, prothrombin time (PT), fibrinogen (Fib), CD4 and CD8 in relation to hepatopulmonary syndrome (HPS) by using a rat model system and to assess the association with pathologic changes in lung. METHODS: Forty male Sprague-Dawley rats were divided into equal groups for modeling of cirrhosis and HPS. The two groups were assessed by blood gas analysis, standard biochemical tests to measure D-dimer, PT, Fib, CD4 and CD8, and pathological examination of lung tissues. RESULTS: The HPS rats showed significantly lower PaO2 than the cirrhosis rats (58.20+/-3.19 mmHg vs. 85.00+/-2.53 mmHg, P = 0.000). The HPS rats showed significantly higher levels of D-dimer, Fib and CD8 than the cirrhosis rats (0.39+/-0.09 mg/ml vs. 0.25+/-0.05 mg/ml, P = 0.000; 1.77+/-0.10 g/L vs. and 1.49+/-0.09 g/L, P = 0.010; 32.32+/-4.45/mm3 vs. 20.13+/-6.09/mm3, P = 0.014). The HPS rats showed significantly lower levels of PT, CD4 and CD4/CD8 than the cirrhosis rats (14.86+/-1.04 s vs. 16.23+/-0.75 s, P = 0.036; 20.45+/-3.86/mm3 vs. 26.75+/-5.32/mm3, P = 0.000; 0.64+/-0.09 vs. 1.32+/-0.13, P = 0.000). The lung tissues of the HPS rats showed microthrombosis in pulmonary vessels, which were not observed in lung tissues of the cirrhosis rats. CONCLUSION: HPS-related differential levels of D-dimer, PT, Fib, CD4, CD8 and CD4/CD8 may represent a biomarker profile suggestive of incidence of thromboembolism in lung.

Annexin A1 protein regulates the expression of PMVEC cytoskeletal proteins in CBDL rat serum-induced pulmonary microvascular remodeling.

BACKGROUND: Hepatopulmonary syndrome (HPS) is characterized by advanced liver disease, hypoxemia and intrapulmonary vascular dilatation (IPVD). The pathogenesis of HPS is not completely understood. Recent findings have established the role of proliferation and phenotype differentiation of pulmonary microvascular endothelial cells (PMVECs) in IPVD of HPS; the change in cytoskeletal proteins and their molecular mechanism play an essential role in the proliferation, phenotype modulation and differentiation of PMVECs. However, little is known about the relevance of cytoskeletal protein expression and its molecular mechanism in IPVD of HPS. In addition, ANX A1 protein has been identified as a key regulator in some important signaling pathways, which influences cytoskeletal remodeling in many diseases, such as lung cancer, liver cancer, etc. METHODS: PMVECs were cultured from the normal rats and then divided into three groups(Ad-ANXA1-transfected group, a non-transfected group, and an adenovirus empty vector group) and incubated by nomal rat serum or hepatopulmonary syndrome rat serum respectively. mRNA level was evaluated by real time reverse transcription polymerase chain reaction, and protein expression was detected by western blot. Cell proliferation was determined by the MTT and thymidine incorporation assay. RESULTS: In this study, we found that the serum from a common bile duct ligation(CBDL) Rat model decreased the expression levels of the ANX A1 mRNA and protein by at least two-fold in human PMVECs. We also found the expression of cytoskeletal proteins (Destrin, a1-actin, and a1-tubulin) in PMVECs significantly increased. After stimulating ANX A1 over-expression in PMVECs by adenovirus-mediated ANX A1 (Ad-ANXA1) transfection, we found the expression levels of cytoskeletal proteins were significantly suppressed in PMVECs at all time points. Additionally, we report here that serum from a CBDL Rat model increases the proliferation of PMVECs by nearly two-fold and that over-expression of Ad-ANXA1 significantly inhibits HPS-rat-serum-induced PMVEC proliferation (p <0.05). These findings suggest that the ANX A1 down-regulation of PMVEC proliferation in the presence of HPS-rat-serum may be the major cause of aberrant dysregulation of cytoskeletal proteins (Destrin, a1-actin, and a1-tubulin) and may, therefore, play a fundamental role in the proliferation and phenotype differentiation of PMVECs in the PVD of HPS. CONCLUSION: Finally, the fact that transfection with Ad-ANXA1 results in inhibition of the aberrant dysregulation of cytoskeletal proteins and proliferation of PMVECs suggests a potential therapeutic effect on PVD of HPS.

[Gaseous disorders and their role in the development of hepatopulmonary syndrome in liver cirrhosis associated with COPD].

OBJECTIVE: To evaluate the indicators of arterial blood gasometry and levels of nitric oxide (NO) in exhaled breath of patients with liver cirrhosis (LC) in combination with COPD, and determine frequency of occurrence of hepatopulmonary syndrome (HPS). The comparison group consisted of 20 patients with COPD, control - 15 volunteers. Gas measurement was estimated by ABL 725 radiometer (Denmark). MATERIAL AND METHODS: We examined 57 patients with liver cirrhosis. The patients were divided into 2 groups: 1st--Patients with liver cirrhosis (n = 48), 2nd group--the CPU and COPD (n = 9). Comparison group consisted of 20 patients with COPD, control - 15 volunteers. The gasometry was evaluated by ABL 725 radiometer (Denmark). NO levels in exhaled air was estimated using the NIOX MINO (Sweden). Contrast electrocardiography was performed on ATL HDI 5000 (Bothell, WA, USA). RESULTS: We found that the PaO2 was lower in the 2nd group compared with the control and with the 1st group (81.4 +/- 5.2; 95.5 +/- 5.3 and 94.5 +/- 5.1 mm Hg; p < 0.05), but the lowest PaO2 was in the group with COPD (68.4 mm Hg). AaDO2 was higher in the group with COPD (37.7 mm Hg). In the 2nd group this indicator was higher as compared with the control and with the 1st group (26.8 +/- 5.4; 8.2 +/- 4.5 and 14.9 +/- 5.2 mm Hg; p < 0.05). A strong negative correlation in the 1st and the 2nd groups between AaDO2 and PaO2 (r = -0.67 and r = -0.93; p < 0.05) was obtained. Content of NO in exhaled air was higher in the 1st and 2nd groups compared with control (18.7 +/- 4.1; 18,9 +/- 4.5 and 11.0 +/- 3.3 ppb; p < 0.05). Intrapulmonary shunts were detected in 3 patients in the 1st and 2nd groups. CONCLUSION: The relationship between measures of blood gas composition showed the leading role of hypoxemia in increasing the risk of formation of HPS in the LC, especially when combined with COPD.

The role of CX3CL1/CX3CR1 in pulmonary angiogenesis and intravascular monocyte accumulation in rat experimental hepatopulmonary syndrome.

BACKGROUND & AIMS: Hepatopulmonary syndrome (HPS), classically attributed to intrapulmonary vascular dilatation, occurs in 15-30% of cirrhotics and causes hypoxemia and increases mortality. In experimental HPS after common bile duct ligation (CBDL), monocytes adhere in the lung vasculature and produce vascular endothelial growth factor (VEGF)-A and angiogenesis ensues and contribute to abnormal gas exchange. However, the mechanisms for these events are unknown. The chemokine fractalkine (CX(3)CL1) can directly mediate monocyte adhesion and activate VEGF-A and angiogenesis via its receptor CX(3)CR1 on monocytes and endothelium during inflammatory angiogenesis. We explored whether pulmonary CX(3)CL1/CX(3)CR1 alterations occur after CBDL and influence pulmonary angiogenesis and HPS. METHODS: Pulmonary CX(3)CL1/CX(3)CR1 expression and localization, CX(3)CL1 signaling pathway activation, monocyte accumulation, and development of angiogenesis and HPS were assessed in 2- and 4-week CBDL animals. The effects of a neutralizing antibody to CX(3)CR1 (anti-CX(3)CR1 Ab) on HPS after CBDL were evaluated. RESULTS: Circulating CX(3)CL1 levels and lung expression of CX(3)CL1 and CX(3)CR1 in intravascular monocytes and microvascular endothelium increased in 2- and 4-week CBDL animals as HPS developed. These events were accompanied by pulmonary angiogenesis, monocyte accumulation, activation of CX(3)CL1 mediated signaling pathways (Akt, ERK) and increased VEGF-A expression and signaling. Anti-CX(3)CR1 Ab treatment reduced monocyte accumulation, decreased lung angiogenesis and improved HPS. These events were accompanied by inhibition of CX(3)CL1 signaling pathways and a reduction in VEGF-A expression and signaling. CONCLUSIONS: Circulating CX(3)CL1 levels and pulmonary CX(3)CL1/CX(3)CR1 expression and signaling increase after CBDL and contribute to pulmonary intravascular monocyte accumulation, angiogenesis and development of experimental HPS.

TNF-α neutralization improves experimental hepatopulmonary syndrome in rats.

BACKGROUND/AIM: TNF-α is increased in hepatopulmonary syndrome (HPS). Pentoxifylline (PTX) mitigated experimental HPS through the inhibition of TNF-α. However, PTX has pleiotropic effects besides the inhibition of TNF-α. This study is to neutralize TNF-α with specific monoclonal antibody to TNF-α (TNF-α McAb) to investigate the effect of TNF-α on HPS. MATERIALS AND METHODS: Hepatopulmonary syndrome was induced by common bile duct ligation (CBDL); controls were sham operated. The endpoints were 1, 2, 3, 4 and 5 weeks after surgery. (99m) Technetium-macroaggregated albumin (Tc-MAA) was to evaluate intrapulmonary arteriovenous shunts; Portal venous pressure, cardiac output and mean blood pressure (MAP) were also measured. Serum was for Alanine transaminase (ALT), endotoxin, TNF-α and nitric oxide (NO) measurements, liver for histology, lung for histology and iNOS, PI3K/Akt expression assay. RESULTS: Portal vein pressure was significantly elevated and MAP decreased in CBDL rats. Tc-MAA was mainly located in lung and very weak in brain in sham group and mainly in brain of CBDL rats. TNF-α McAb significantly decreased the radioactivity in the brain, reduced cardiac output, increased MAP and systemic vascular resistance (SVR) in CBDL animals. Serum ALT, endotoxin, TNF-α and NO were significantly increased. TNF-α McAb significantly decreased these serum indices in CBDL rats. TNF-α McAb significantly alleviated liver damage, decreased alveolar-arterial gradient and inhibited iNOS, PI3K/Akt and p-Akt expression in lung tissue. Furthermore, TNF-α McAb significantly attenuated the inflammatory response in lung. CONCLUSION: TNF-α McAb improves HPS in cirrhotic rats; this effect is likely mediated through the inhibition of TNF-α PI3K/Akt-NO pathway.

Elevated levels of endothelin-1 in hepatic venous blood are associated with intrapulmonary vasodilatation in humans.

BACKGROUND: Hepatopulmonary syndrome is a pulmonary vascular complication of cirrhosis in which intrapulmonary vasodilatation (IPV) results in hypoxemia. Endothelin-1 (ET-1), produced by proliferating cholangiocytes, has been identified as a mediator of IPV in an animal model of HPS, but the pathophysiology of IPV in humans has not been defined. AIM: The purpose of this study was to assess whether cirrhosis with IPV, which often leads to HPS, is associated with increased hepatic venous ET-1 blood levels. METHODS: We performed a prospective cohort pilot study of 40 patients with liver disease undergoing transjugular liver biopsy from November 1, 2008 to September 1, 2009. Patients were categorized according to absence (-) or presence (+) of IPV as determined by bubble-contrasted echocardiography. Hepatic venous blood was assayed for ET-1 by ELISA. The percent volume of cholangiocytes in the liver biopsy specimen was determined by morphometric analysis, as a measure of bile duct proliferation. RESULTS: Nine subjects were excluded, due to absence of cirrhosis (6) and patent foramen ovale (3). Of the remaining 31 subjects, IPV was present in 18 (58%). Median hepatic venous ET-1 was higher with IPV+ than IPV- at levels of 9.1 pg/mL (range 7.5-11.7) versus 2.1 pg/mL (1.3-5.6), respectively (P = 0.004). ET-1 levels correlated positively with cholangiocyte percent volume (r = 0.72, P < 0.001) but not with measures of liver dysfunction (bilirubin, INR, MELD score, or hepatic venous pressure gradient). CONCLUSION: In human cirrhosis, increased hepatic venous ET-1 is associated with IPV and increased hepatic cholangiocyte volume.

Reduced circulating BMP10 and BMP9 and elevated endoglin are associated with disease severity, decompensation and pulmonary vascular syndromes in patients with cirrhosis.

BACKGROUND: BMP9, originating from the liver, and BMP10 are circulating BMPs that preserve vascular endothelial integrity. We assessed BMP9, BMP10 and soluble endoglin (sEng) levels and their relationships to liver disease severity and associated pulmonary vascular syndromes in a cohort of well-characterised liver disease patients. METHODS: Plasma samples from patients with liver disease (n = 83) and non-disease controls (n = 21) were assayed for BMP9, BMP10 and sEng. Levels were also assessed in a separate cohort of controls (n = 27) and PoPH patients (n = 8). Expression of mRNA and immunohistochemical staining was undertaken in liver biopsy specimens. Plasma BMP activity was assessed using an endothelial cell bioassay. FINDINGS: Plasma BMP9 and BMP10 levels were normal in patients with compensated cirrhosis or fibrosis without cirrhosis, but markedly reduced in patients with decompensated cirrhosis, including those with hepatopulmonary syndrome (HPS) or portopulmonary hypertension (PoPH). Liver biopsy specimens revealed reduced mRNA expression and immunostaining for these ligands. Patient plasma samples with reduced BMP9 and BMP10 levels exhibited low BMP activity that was restored with exogenous BMP9. Endoglin mRNA expression was increased in cirrhotic livers and elevated circulating sEng levels in PoPH and HPS patients suggested increased endothelial sEng shedding in these syndromes. INTERPRETATION: Plasma BMP9 and BMP10 levels are reduced in decompensated cirrhosis, leading to reduced circulating BMP activity on the vascular endothelium. The pulmonary complications of cirrhosis, PoPH and HPS, are associated with markedly reduced BMP9 and BMP10 and increased sEng levels, suggesting that supplementation with exogenous ligands might be a therapeutic approach for PoPH and HPS.

Pulmonary vascular complications in asymptomatic children with portal hypertension.

OBJECTIVES: : To determine the prevalence of portopulmonary hypertension, hepatopulmonary syndrome (HPS), and intrapulmonary vascular shunting (IPVS) in children with clinically stable portal hypertension and to assess the value of vasoactive peptide levels, biochemical tests and clinical signs or symptoms to predict these conditions. PATIENTS AND METHODS: : A prospective, cross-sectional analysis was conducted on 33 children, ages 4 to 17 years, with stable cirrhosis (n = 28) or extrahepatic portal hypertension (n = 5). The children were screened for IPVS and hypoxia with contrast-enhanced echocardiography (cECHO) and pulse oximetry, and screened for pulmonary hypertension with Doppler echocardiography. Chemistries, radiographs, physical examinations, and levels of vasoactive peptides were compared between subjects with IPVS and those with normal cECHO. RESULTS: : No subject had pulmonary hypertension. Six (19%) had IPVS, all of which had intrahepatic causes of portal hypertension, and 1 of whom had HPS. Compared with subjects with normal cECHO, those with IPVS had biochemical evidence of more advanced liver disease and higher b-type natriuretic peptide levels. CONCLUSIONS: : Prevalence of portopulmonary hypertension and HPS appear to be rare in clinically stable children with portal hypertension. Intrapulmonary vascular shunting was present in 19% of these patients. A novel finding of this study is the elevation of b-type natriuretic peptide in children with IPVS.