CT Scan Does Not Differentiate Patients with Hepatopulmonary Syndrome from Other Patients with Liver Disease

<div><p>Background</p><p>Hepatopulmonary syndrome (HPS) is defined by liver dysfunction, intrapulmonary vascular dilatations, and impaired oxygenation. The gold standard for detection of intrapulmonary vascular dilatations in HPS is contrast echocardiography. However, two small studies have suggested that patients with HPS have larger segmental pulmonary arterial diameters than both normal subjects and normoxemic subjects with cirrhosis, when measured by CT. We sought to compare CT imaging-based pulmonary vasodilatation in patients with HPS, patients with liver dysfunction without HPS, and matching controls on CT imaging.</p><p>Methods</p><p>We performed a retrospective cohort study at two quaternary care Canadian HPS centers. We analyzed CT thorax scans in 23 patients with HPS, 29 patients with liver dysfunction without HPS, and 52 gender- and age-matched controls. We measured the artery-bronchus ratios (ABRs) in upper and lower lung zones, calculated the “delta ABR” by subtracting the upper from the lower ABR, compared these measurements between groups, and correlated them with clinically relevant parameters (partial pressure of arterial oxygen, alveolar-arterial oxygen gradient, macroaggregated albumin shunt fraction, and diffusion capacity). We repeated measurements in patients with post-transplant CTs.</p><p>Results</p><p>Patients had significantly larger lower zone ABRs and delta ABRs than controls (1.20 +/- 0.19 versus 0.98 +/- 0.10, p<0.01; and 0.12 +/- 0.17 versus -0.06 +/- 0.10, p<0.01, respectively). However, there were no significant differences between liver disease patients with and without HPS, nor any significant correlations between CT measurements and clinically relevant parameters. There were no significant changes in ABRs after liver transplantation (14 patients).</p><p>Conclusions</p><p>Basilar segmental artery-bronchus ratios are larger in patients with liver disease than in normal controls, but this vasodilatation is no more severe in patients with HPS. CT does not distinguish patients with HPS from those with uncomplicated liver disease.</p></div

Patient and Control CT Measurements.

<p>Patient and Control CT Measurements.</p

Axial CT images of the right lower zone in a patient with hepatopulmonary syndrome (A), subclinical hepatopulmonary syndrome (B), liver dysfunction only (C), and control subject (D) with representative artery and accompanying bronchus highlighted (brackets).

<p>Axial CT images of the right lower zone in a patient with hepatopulmonary syndrome (A), subclinical hepatopulmonary syndrome (B), liver dysfunction only (C), and control subject (D) with representative artery and accompanying bronchus highlighted (brackets).</p

CT scans from a control subject demonstrating bronchovascular measurements.

<p>Axial images demonstrate measurements of the main pulmonary artery (A), the right pulmonary artery (B), and the left pulmonary artery (C). Coronal image from the same subject demonstrates location of the upper and lower zones for bronchovascular measurements (brackets) (D).</p

Pharmacological characterization of the receptor involved in the protective effect of ODN on astroglial cells.

<p>(A) Cultured astrocytes were pre-incubated for 30 min in the absence or presence of ODN (0.1 nM), the metabotropic receptor agonist OP (0.1 nM), the inactive ODN analog [Ala¹⁵]ODN (0.1 nM) or the specific CBR agonist clonazepam (Clona; 10 nM), and then incubated for 1 h with medium alone or with 300 µM H₂O₂ without or with receptor ligands. (B) Cells were pre-incubated for 30 min in the absence or presence of the metabotropic receptor antagonist cyclo_1–8 [DLeu⁵] OP (c[DLeu⁵] OP; 1 µM) or the CBR antagonist flumazenil (1 µM), and then incubated for 1 h with medium alone or with 300 µM H₂O₂ without or with ODN (0.1 nM). Cell survival was quantified by measuring FDA fluorescence intensity, and the results are expressed as percentages of control. Each value is the mean (± SEM) of at least 12 different wells from three independent cultures. ANOVA followed by the Bonferroni's test. <i>*** p</i><0.001; NS, not statistically different <i>vs.</i> control. ^###<i>p</i><0.001; ns, not statistically different <i>vs.</i> H₂O₂-treated cells.</p

Involvement of the mitochondrial intrinsic pathway in the protective effect of ODN on astroglial cells.

<p>(A) Cultured astrocytes were pre-incubated for 10 min in the absence (a, c) or presence of 0.1 nM ODN (b, d), and then incubated for 1 h with medium alone (a), ODN (b) or with 300 µM H₂O₂ without (c) or with ODN (d). Aggregated (red signal) and monomeric (green signal) fluorescent JC-1 dye revealed active and inactive mitochondria, respectively. Scale bar, 50 µm. (B) Cultured astrocytes were pre-incubated for 10 min in the absence or presence of 0.1 nM ODN, and then incubated for 1 h with medium alone, ODN or with 300 µM H₂O₂ without or with ODN. Bcl-2 and Bax mRNA levels were quantified by RT-PCR. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042498#s2" target="_blank">Results</a> are expressed as percentages of control. Each value is the mean (± SEM) of 6 different wells from three independent cultures. ANOVA followed by the Bonferroni's test. * <i>p</i><0.05; NS, not statistically different <i>vs.</i> control. ^#<i>p</i><0.05; ^##<i>p</i><0.01 <i>vs.</i> H₂O₂-treated cells. (C) Cultured astrocytes were pre-incubated for 30 min in the absence or presence of H89 (20 µM), U0126 (20 µM), U73122 (1 µM) or chelerythrine (Chel; 1 µM) and then incubated for 1 h with medium alone, ODN (0.1 nM), dbcAMP (1 mM) or with 300 µM H₂O₂ without or with ODN (0.1 nM) or dbcAMP (1 mM). Caspase-3 activity was quantified by measuring the fluorescence of caspase substrate, Z-DEVD-Rhodamine 110, and the results are expressed as percentages of control. Each value is the mean (± SEM) of at least 12 different wells from three independent cultures. ANOVA followed by the Bonferroni's test. <i>*** p</i><0.001; NS, not statistically different <i>vs.</i> control. ^###<i>p</i><0.001; ns, not statistically different <i>vs.</i> H₂O₂-treated cells.</p

Schematic representation of the signaling pathways likely involved in the protective effect of ODN against H₂O₂-induced astroglial cell apoptosis.

<p>ODN activates both adenylyl cyclase (AC) and phospholipase C (PLC) in astrocytes, however only the cAMP-dependent protein kinase A (PKA) pathway is involved in the effect of ODN on cell survival. ODN stimulates phosphorylation of extracellular regulated kinase (ERK) in a PKA-dependent manner. Downstream, ODN activates the expression of the anti-apoptotic gene Bcl-2, stimulates glutathione (GSH) formation and abolishes H₂O₂-induced decrease of mitochondrial potential (Ψm) via the formation of highly reactive oxygen species (ROS), the inhibition of Bcl-2 and the stimulation of the pro-apoptotic gene Bax. Finally, ODN prevents H₂O₂-evoked activation of caspase-3 leading to astrocyte death. Cyt c, cytochrome c; DAG, diacylglycerol; IP₃, inositol trisphosphate; H89, protein kinase A inhibitor; MEK, mitogen-activated protein kinase kinase; PKC, protein kinase C; U0126, MAP kinase kinase inhibitor. +, activation; –, inhibition.</p

Protective effect of ODN on astroglial cell death induced by H₂O₂.

<p>(A) Cultured astrocytes were pre-incubated for 10 min in the absence or presence of graded concentrations of ODN (1 fM–1 nM) and then incubated for 1 h with medium alone (□) or with 300 µM H₂O₂ without (•) or with ODN (▪). Cell survival was quantified by measuring FDA fluorescence intensity, and the results are expressed as percentages of control. Each value is the mean (± SEM) of at least 12 different wells from three independent cultures. ANOVA followed by the Bonferroni's test: <i>** p</i><0.01; <i>*** p</i><0.001; NS, not statistically different <i>vs.</i> control. ^#<i>p</i><0.05; ^###<i>p</i><0.01; ns, not statistically different <i>vs.</i> H₂O₂-treated cells. (B) Typical images illustrating the protective effect of ODN on H₂O₂-induced cell death. Cells were pre-incubated for 10 min in the absence (a, c) or presence of 0.1 nM ODN (b, d), and then incubated for 1 h with medium alone (a), ODN (b) or with 300 µM H₂O₂ without (c) or with ODN (d). Living astrocytes were labeled with calcein-AM (green fluorescence staining). Scale bar = 50 µm. (C) Time-course effect of ODN on H₂O₂-induced cell death. Cells were incubated for the indicated times with medium alone (□) or 300 µM H₂O₂ without (•) or with ODN (0.1 nM; ▪). After 30, 60, 90, 120 or 150 min of incubation, a refill of 0.1 nM ODN (arrows; ▴) was added in the culture medium. Cell survival was quantified by measuring FDA fluorescence intensity, and the results are expressed as percentages of control. Each value is the mean (± SEM) of at least 12 different wells from three independent cultures. ANOVA followed by the Bonferroni's test. ^##<i>p</i><0.01; ^###<i>p</i><0.001 <i>vs.</i> H₂O₂-treated cells.</p

Identification of intracellular pathways involved in the protective effect of ODN on astroglial cells.

<p>(A) Cultured astrocytes were pre-incubated for 30 min in the absence or presence of H89 (20 µM), U0126 (20 µM), U73122 (1 µM) and chelerythrine (Chel; 1 µM) and then incubated for 1 h with medium alone, ODN (0.1 nM), or with 300 µM H₂O₂ without or with ODN (0.1 nM). (B) Cultured astrocytes were pre-incubated for 30 min in the absence or presence of the PKA inhibitor H89 (20 µM) and the ERK inhibitors U0126 (20 µM), and then incubated for 1 h with medium alone or with 300 µM H₂O₂ without or with dbcAMP (1 mM). Cell survival was quantified by measuring FDA fluorescence intensity, and the results are expressed as percentages of the control. Each value is the mean (± SEM) of at least of 12 different wells from three independent cultures. ANOVA followed by the Bonferroni's test. <i>*** p</i><0.001; NS, not statistically different <i>vs.</i> control. ^###<i>p</i><0.001; ns, not statistically different <i>vs.</i> H₂O₂-treated cells.</p

Effect of ODN on cAMP formation in astroglial cells.

<p>(A) Cultured astrocytes were pre-incubated for 30 min in the presence of 0.1 mM IBMX, and then incubated for 10 min with graded concentrations of ODN (1 fM–10 nM). (B) Cultured astrocytes were pre-incubated for 30 min with 0.1 mM IBMX in the absence or presence of cyclo_1–8 [DLeu⁵] OP (c[DLeu⁵] OP; 1 µM) or flumazenil (1 µM), and then incubated for 10 min with medium alone or with ODN (0.1 nM). The results are expressed as percentages of control. cAMP production was quantified by radioimmunoassay, and the results are expressed as percentages of control. Each value is the mean (± SEM) of at least 15 different wells from three independent cultures. ANOVA followed by the Bonferroni's test. <i>* p</i><0.05; <i>** p</i><0.01; <i>*** p</i><0.001; NS, not statistically different <i>vs.</i> control. ^###<i>p</i><0.001; ns, not statistically different <i>vs.</i> cells incubated with antagonists.</p