Liver International ISSN 1478-3223
ORIGINAL ARTICLE
Role of angiogenic factors/cell adhesion markers in serum of cirrhotic patients with hepatopulmonary syndrome Sarah Raevens1, Stephanie Coulon1, Christophe Van Steenkiste1,2, Roos Colman3, Xavier Verhelst1, Hans Van Vlierberghe1, Anja Geerts1, Thomas Perkmann4, Thomas Horvatits5, Valentin Fuhrmann5 and Isabelle Colle6 1 2 3 4 5 6
Department of Gastroenterology and Hepatology, Ghent University Hospital, Ghent, Belgium Department of Gastroenterology and Hepatology, Maria Middelares Hospital, Ghent, Belgium Department of Public Health, Ghent University, Ghent, Belgium Department of Laboratory Medicine, Medical University Vienna, Vienna, Austria Department of Gastroenterology and Hepatology, Medical University Vienna, Vienna, Austria Department of Gastroenterology and Hepatology, Algemeen Stedelijk Ziekenhuis ASZ, Aalst, Belgium and Ghent University, Ghent, Belgium
Keywords angiogenesis – cirrhosis – hepatopulmonary syndrome – ICAM3 – VCAM1 Abbreviations AIC, Akaike information criterion; AUC, area under the curve; bFGF, basic fibroblast growth factor; cKit, tyrosine-protein kinase kit; CO, carbon monoxide; CRP, C-reactive protein; CX3CL1, fractalkine; eNOS, endothelial nitric oxide synthase; ERS, European Respiratory Society; FEV1, forced expiratory volume in 1 second; HIF, hypoxiainducable factor; HPS, hepatopulmonary syndrome; ICAM1, intercellular adhesion molecule 1; ICAM3, intercellular adhesion molecule 3; IL, interleukin; iNOS, inducible nitric oxide synthase; IPVD, intrapulmonary vascular dilations; LFA-1, lymphocyte function-associated antigen; LR+, positive likelihood ratio; LR, negative likelihood ratio; NO, nitric oxide; NPV, negative predictive value; P(A-a) O2 gradient, room air alveolar– arterial oxygen gradient; PlGF, placental growth factor; PPV, positive predictive value; ROC curve, receiver operating characteristic curve; SAA, serum amyloid A; sFlt1, soluble fms-like tyrosine kinase-1 or svegfr-1; Throm, thrombomodulin; Tie2, angiopoietin receptor TEK tyrosine kinase; TNFa, tumour necrosis factor alpha; TTE, transthoracic echocardiography; VCAM1, vascular cell adhesion molecule 1; VC, vital capacity; VEGF-C, vascular endothelial growth factor C; VEGF-D, vascular endothelial growth factor D; VEGFR 1, vascular endothelial growth factor 1; VEGFR 2, vascular endothelial growth factor 2; VEGF, vascular endothelial growth factor A.
Abstract 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 (1) to identify angiogenic factors in serum of patients with hepatopulmonary syndrome and (2) 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. Correspondence Isabelle Colle, MD, PhD, Department of Gastroenterology and Hepatology Algemeen Stedelijk Ziekenhuis ASZ, Aalst, Belgium and Ghent University, Ghent, Belgium. Tel: 0032 53 76 66 40 Fax: 0032 53 76 66 41 e-mail: [email protected]
Received 28 February 2014 Accepted 22 April 2014 DOI:10.1111/liv.12579
Liver International (2014) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
1
Raevens et al.
Role of cell adhesion markers in HPS
The hepatopulmonary syndrome (HPS) refers to a decreased systemic arterial oxygenation because of intrapulmonary vascular dilations (IPVD) that can occur in patients with acute and chronic liver disease (1, 2). The prevalence of HPS is estimated to range from 4 to 47% among patients with chronic liver disease (3– 6). Clinical criteria for HPS consist of the following triad: (i) an elevated room air alveolar–arterial oxygen gradient (P(A-a) O2 gradient >15 mmHg or >20 mmHg when age older than 65 years), (ii) the presence of liver disease and (iii) evidence of IPVD, especially in the basal parts of the lungs (1). Hepatopulmonary syndrome results in a significant increase in morbidity and mortality. The hypoxaemia is generally progressive and mortality is significantly increased. Without liver transplantation, a mean survival of approximately 24 months can be expected (1). Studies have shown a 5-year survival rate of 23% (7). Diagnosis of HPS consists of demonstrating the presence of impaired gas exchange and IPVDs. An increased P(Aa) O2 gradient is documented with arterial blood gas in upright position. The presence of IPVDs can be documented by contrast transthoracic echocardiography (TTE), which is the gold standard, or nuclear lung scanning (8). In conclusion, diagnostic criteria for HPS include a PaO2 less than 80 mmHg or a P (A-a) O2 gradient more than 15 mmHg on room air, evidence of IPVDs on contrast-enhanced echocardiography or nuclear scanning, in the setting of liver disease, portal hypertension with or without cirrhosis. The presence of IPVD is considered as the main cause of hypoxaemia in HPS. A combination of pulmonary vasodilation and pulmonary capillary proliferation of arteriovenous malformations leads to these IPVD. An imbalance between vasodilators and vasoconstrictors plays an important role. In recent literature, evidence mounts for a process of angiogenesis. Angiogenesis is a recurring factor seen in the pathophysiology of chronic liver disease and its complications such as portal hypertension, cirrhosis, non-alcoholic steatohepatitis and HCC (9). A strong association between inflammation and angiogenesis has been confirmed in several studies (10). Inflammation triggers vascular permeability by recruiting leucocytes, which are able to initiate angiogenesis in different manners. Pro-inflammatory cytokines released during inflammation can trigger the angiogenic process by induction of hypoxia and hypoxiainducible factors (HIF) (9). In HPS, intestinal bacterial translocation and intestinal endotoxaemia with increased circulation of TNFa and monocytes adherence in the lung vasculature were studied, with downstream production of vascular endothelial growth factor A (VEGF-A) and angiogenesis ensues. This triggers activation of Akt and endothelial nitric oxide synthase (eNOS) leading to abnormal gas exchange. The fact that there is a close relationship between liver disease and its complications, of one is
2
HPS, and angiogenesis brings about the goal to detect pro-angiogenic factors that may be a noninvasive way to identify patients with HPS. In this study, possible angiogenic factors in serum of patients with HPS were identified and the possibility to predict the presence or absence of HPS by these angiogenic factors was studied. Patients and methods Patients
A total of 60 patients with cirrhosis, including inpatients and outpatients, of whom 30 with HPS and 30 without HPS, were studied. All patients were screened for the presence of HPS and had blood sampling at the Medical University Vienna, Austria. Patients in HPS and no HPS groups were matched for severity and aetiology of liver disease. Patients with lung disease were excluded from this study, as several lung conditions may elevate VCAM and ICAM levels and may lead to an increased P(A-a) O2 gradient. All patients were tested by spirometry with determination of the forced expiratory volume in 1 second (FEV1), vital capacity (VC) and calculation of the FEV1/VC ratio or Tiffeneau index. All patients had normal lung functions with a FEV1 normal for age and sex and a FEV1/VD index >70%, thereby ruling out obstructive lung diseases such as asthma, chronic obstructive lung disease, chronic bronchitis and emphysema. There was no exclusion of current or ex-smoking. All patients signed an informed consent before entering this study. Before blood sampling, the control subjects signed a written informed consent. The procedures were approved by the ethics committee of the Medical University Vienna. Diagnosis of hepatopulmonary syndrome
Hepatopulmonary syndrome was defined by (i) positive contrast-enhanced transthoracic echocardiography (TTE, detection of IPVD), (ii) presence of cirrhosis and (iii) a P(A-a) O2 gradient ≥15 mmHg (or ≥20 mmHg in patients aged >64 years) and arterial oxygen tension (PaO2) 10 lm in diameter that usually do not pass through the pulmonary capillary bed. Appearance of microbubbles, after injecting in a peripheral vein, first in the right heart, and within 3–6 heart actions in the left heart demonstrates abnormal vasodilation of the intrapulmonary capillary bed. Early (
ORIGINAL ARTICLE
Role of angiogenic factors/cell adhesion markers in serum of cirrhotic patients with hepatopulmonary syndrome Sarah Raevens1, Stephanie Coulon1, Christophe Van Steenkiste1,2, Roos Colman3, Xavier Verhelst1, Hans Van Vlierberghe1, Anja Geerts1, Thomas Perkmann4, Thomas Horvatits5, Valentin Fuhrmann5 and Isabelle Colle6 1 2 3 4 5 6
Department of Gastroenterology and Hepatology, Ghent University Hospital, Ghent, Belgium Department of Gastroenterology and Hepatology, Maria Middelares Hospital, Ghent, Belgium Department of Public Health, Ghent University, Ghent, Belgium Department of Laboratory Medicine, Medical University Vienna, Vienna, Austria Department of Gastroenterology and Hepatology, Medical University Vienna, Vienna, Austria Department of Gastroenterology and Hepatology, Algemeen Stedelijk Ziekenhuis ASZ, Aalst, Belgium and Ghent University, Ghent, Belgium
Keywords angiogenesis – cirrhosis – hepatopulmonary syndrome – ICAM3 – VCAM1 Abbreviations AIC, Akaike information criterion; AUC, area under the curve; bFGF, basic fibroblast growth factor; cKit, tyrosine-protein kinase kit; CO, carbon monoxide; CRP, C-reactive protein; CX3CL1, fractalkine; eNOS, endothelial nitric oxide synthase; ERS, European Respiratory Society; FEV1, forced expiratory volume in 1 second; HIF, hypoxiainducable factor; HPS, hepatopulmonary syndrome; ICAM1, intercellular adhesion molecule 1; ICAM3, intercellular adhesion molecule 3; IL, interleukin; iNOS, inducible nitric oxide synthase; IPVD, intrapulmonary vascular dilations; LFA-1, lymphocyte function-associated antigen; LR+, positive likelihood ratio; LR, negative likelihood ratio; NO, nitric oxide; NPV, negative predictive value; P(A-a) O2 gradient, room air alveolar– arterial oxygen gradient; PlGF, placental growth factor; PPV, positive predictive value; ROC curve, receiver operating characteristic curve; SAA, serum amyloid A; sFlt1, soluble fms-like tyrosine kinase-1 or svegfr-1; Throm, thrombomodulin; Tie2, angiopoietin receptor TEK tyrosine kinase; TNFa, tumour necrosis factor alpha; TTE, transthoracic echocardiography; VCAM1, vascular cell adhesion molecule 1; VC, vital capacity; VEGF-C, vascular endothelial growth factor C; VEGF-D, vascular endothelial growth factor D; VEGFR 1, vascular endothelial growth factor 1; VEGFR 2, vascular endothelial growth factor 2; VEGF, vascular endothelial growth factor A.
Abstract 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 (1) to identify angiogenic factors in serum of patients with hepatopulmonary syndrome and (2) 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. Correspondence Isabelle Colle, MD, PhD, Department of Gastroenterology and Hepatology Algemeen Stedelijk Ziekenhuis ASZ, Aalst, Belgium and Ghent University, Ghent, Belgium. Tel: 0032 53 76 66 40 Fax: 0032 53 76 66 41 e-mail: [email protected]
Received 28 February 2014 Accepted 22 April 2014 DOI:10.1111/liv.12579
Liver International (2014) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
1
Raevens et al.
Role of cell adhesion markers in HPS
The hepatopulmonary syndrome (HPS) refers to a decreased systemic arterial oxygenation because of intrapulmonary vascular dilations (IPVD) that can occur in patients with acute and chronic liver disease (1, 2). The prevalence of HPS is estimated to range from 4 to 47% among patients with chronic liver disease (3– 6). Clinical criteria for HPS consist of the following triad: (i) an elevated room air alveolar–arterial oxygen gradient (P(A-a) O2 gradient >15 mmHg or >20 mmHg when age older than 65 years), (ii) the presence of liver disease and (iii) evidence of IPVD, especially in the basal parts of the lungs (1). Hepatopulmonary syndrome results in a significant increase in morbidity and mortality. The hypoxaemia is generally progressive and mortality is significantly increased. Without liver transplantation, a mean survival of approximately 24 months can be expected (1). Studies have shown a 5-year survival rate of 23% (7). Diagnosis of HPS consists of demonstrating the presence of impaired gas exchange and IPVDs. An increased P(Aa) O2 gradient is documented with arterial blood gas in upright position. The presence of IPVDs can be documented by contrast transthoracic echocardiography (TTE), which is the gold standard, or nuclear lung scanning (8). In conclusion, diagnostic criteria for HPS include a PaO2 less than 80 mmHg or a P (A-a) O2 gradient more than 15 mmHg on room air, evidence of IPVDs on contrast-enhanced echocardiography or nuclear scanning, in the setting of liver disease, portal hypertension with or without cirrhosis. The presence of IPVD is considered as the main cause of hypoxaemia in HPS. A combination of pulmonary vasodilation and pulmonary capillary proliferation of arteriovenous malformations leads to these IPVD. An imbalance between vasodilators and vasoconstrictors plays an important role. In recent literature, evidence mounts for a process of angiogenesis. Angiogenesis is a recurring factor seen in the pathophysiology of chronic liver disease and its complications such as portal hypertension, cirrhosis, non-alcoholic steatohepatitis and HCC (9). A strong association between inflammation and angiogenesis has been confirmed in several studies (10). Inflammation triggers vascular permeability by recruiting leucocytes, which are able to initiate angiogenesis in different manners. Pro-inflammatory cytokines released during inflammation can trigger the angiogenic process by induction of hypoxia and hypoxiainducible factors (HIF) (9). In HPS, intestinal bacterial translocation and intestinal endotoxaemia with increased circulation of TNFa and monocytes adherence in the lung vasculature were studied, with downstream production of vascular endothelial growth factor A (VEGF-A) and angiogenesis ensues. This triggers activation of Akt and endothelial nitric oxide synthase (eNOS) leading to abnormal gas exchange. The fact that there is a close relationship between liver disease and its complications, of one is
2
HPS, and angiogenesis brings about the goal to detect pro-angiogenic factors that may be a noninvasive way to identify patients with HPS. In this study, possible angiogenic factors in serum of patients with HPS were identified and the possibility to predict the presence or absence of HPS by these angiogenic factors was studied. Patients and methods Patients
A total of 60 patients with cirrhosis, including inpatients and outpatients, of whom 30 with HPS and 30 without HPS, were studied. All patients were screened for the presence of HPS and had blood sampling at the Medical University Vienna, Austria. Patients in HPS and no HPS groups were matched for severity and aetiology of liver disease. Patients with lung disease were excluded from this study, as several lung conditions may elevate VCAM and ICAM levels and may lead to an increased P(A-a) O2 gradient. All patients were tested by spirometry with determination of the forced expiratory volume in 1 second (FEV1), vital capacity (VC) and calculation of the FEV1/VC ratio or Tiffeneau index. All patients had normal lung functions with a FEV1 normal for age and sex and a FEV1/VD index >70%, thereby ruling out obstructive lung diseases such as asthma, chronic obstructive lung disease, chronic bronchitis and emphysema. There was no exclusion of current or ex-smoking. All patients signed an informed consent before entering this study. Before blood sampling, the control subjects signed a written informed consent. The procedures were approved by the ethics committee of the Medical University Vienna. Diagnosis of hepatopulmonary syndrome
Hepatopulmonary syndrome was defined by (i) positive contrast-enhanced transthoracic echocardiography (TTE, detection of IPVD), (ii) presence of cirrhosis and (iii) a P(A-a) O2 gradient ≥15 mmHg (or ≥20 mmHg in patients aged >64 years) and arterial oxygen tension (PaO2) 10 lm in diameter that usually do not pass through the pulmonary capillary bed. Appearance of microbubbles, after injecting in a peripheral vein, first in the right heart, and within 3–6 heart actions in the left heart demonstrates abnormal vasodilation of the intrapulmonary capillary bed. Early (
