Plasma angiopoietin 2 as a novel prognostic biomarker in alcohol-related cirrhosis and hepatitis

Abstract

Background and aim: Severe alcoholic hepatitis (SAH), the most florid form of alcohol-related liver disease (ALD), has a mortality rate of 16% at 28 days. The angiopoietin-Tie 2 system regulates angiogenesis and inflammation, both of which are implicated in the pathogenesis of ALD. This study examined plasma and hepatic gene expression of angiopoietin 1 (ANG1) and angiopoietin 2 (ANG2) in patients with SAH and ALD and investigated their roles as prognostic biomarkers.

Methods: A case-control study was performed measuring plasma levels of ANG1 and ANG2 by enzyme-linked immunosorbent assay (ELISA) from 30 patients with SAH (Maddrey's discriminant function ≥32), 32 patients with ALD cirrhosis and 15 healthy controls (HC). RNA sequencing for ANG1, ANG2, TIE1 (codes for Tie1 receptor) and TEK (codes for Tie2 receptor) gene expression from a separate cohort study of 79 patients was also performed.

Results: Plasma levels of ANG1 were lower (P = 0.010) and ANG2 were higher (P < 0.0001) in patients with ALD/SAH compared to HC. The ANG2: ANG1 ratio was higher in those with ALD/SAH compared to HC (P < 0.0001). ANG2 levels were the highest in patients who developed sepsis (P = 0.030) and those dying within 90 days (P = 0.020). ANG2 levels correlated positively with model for end-stage liver disease (MELD) score (r = 0.30, P = 0.020), Child-Pugh score (r = 0.38, P = 0.003), international normalized ratio (r = 0.41, P = 0.001) and white blood cell count (r = 0.28, P = 0.040) and inversely correlated with albumin (r = -0.26, P = 0.040).ANG1 gene expression from liver biopsies was higher in SAH than that in HC (P < 0.0001), and greater in severe disease (P < 0.0001). ANG2 gene expression trended towards being lower in SAH than that in HC (P = 0.070) though was upregulated in severe disease (P = 0.0003).

Conclusions: Plasma ANG2 is raised in SAH and ALD and could be useful as a prognostic biomarker in this patient population.

Keywords: Alcohol-related cirrhosis; Alcohol-related liver disease (ALD); Alcoholic hepatitis; Angiopoietin 1 (ANG1); Angiopoietin 2 (ANG2).

Fig. 1

Kaplan–Meier survival curve of all-cause mortality for all SAH patients and ALD cirrhosis patients at 90 days (log rank Mantel-CoxP= 0.020). Abbreviations: ALD, alcohol-related liver disease; SAH, severe alcoholic hepatitis.

Fig. 2

Plasma ANG levels in HC and patients (SAH and ALD cirrhosis). Plasma (A) ANG1 and (B) ANG2 levels in HC and patients. (C) Plasma ANG2: ANG1 ratio in HC and patients. (D) Plasma ANG2 levels in non-septic and septic patients. (E) Plasma ANG2 levels in surviving and non-surviving patients at 90 days. For box-and-whisker plots: midline, median; perimeters, 25^th to 75^th centile; whiskers, minimum to maximum values. The Mann–Whitney U test was used for statistical analysis. ∗P < 0.05, ∗∗∗∗P < 0.0001. Abbreviations: ALD, alcohol-related liver disease; ANG, angiopoietin; HC, healthy control; SAH, severe alcoholic hepatitis.

Fig. 3

ROC analysis showing the discriminatory power of ANG2 between surviving and non-surviving patients at 90 days(AUC 0.72;95% CI: 0.59–0.85;P= 0.020). Abbreviations: ANG, angiopoietin; AUC, area under the curve; CI, confidence interval; ROC, receiver operating characteristic.

Fig. 4

Correlation between plasma ANG2 levels and liver disease severity scores and biochemical markers of severity.(A) Correlation between MELD score and ANG2 levels in patient group (r = 0.30, P = 0.020). (B) Correlation between Child-Pugh score and ANG2 levels in patient group (r = 0.38, P = 0.003). (C) Correlation between INR and ANG2 levels in patient group (r = 0.41, P = 0.001). (D) Correlation between albumin and ANG2 levels in patient group (r = −0.26, P = 0.040). Spearman's rank correlation test was used for statistical analysis. One patient (SAH) was not included in analysis for MELD score, Child-Pugh score and albumin due to missing data. Abbreviations: ANG, angiopoietin; INR, international normalized ratio; MELD, model for end-stage liver disease.

Fig. 5

ANG1RNA andANG2RNA results.(A)ANG1 expression in all groups. (B)ANG2 expression in all groups. Gene expression levels are presented as transcripts per million (tpm). For box-and-whisker plots: midline, median; perimeters, 25^th to 75^th centile; whiskers, minimum to maximum values. The Kruskal-Wallis test and Dunn's multiple comparison test were used for statistical analysis. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. Abbreviations: AH, alcoholic hepatitis; ANG, angiopoietin; exAH, explants from patients with SAH who underwent transplantation; HC, healthy control; HCV, non cirrhotic HCV infection group; HCV-C, compensated HCV-related cirrhosis group; NAFLD, non-alcoholic fatty liver disease; RNA, ribonucleic acid; SAH, severe alcoholic hepatitis.

Fig. 6

TIE1RNA andTEKRNA results.(A)TIE1 expression in all groups. (B)TEK expression in all groups. Gene expression levels are presented as transcripts per million (tpm). For box-and-whisker plots: midline, median; perimeters, 25^th to 75^th centile; whiskers, minimum to maximum values. The Kruskal–Wallis test and Dunn's multiple comparison test were used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Abbreviations: AH, alcoholic hepatitis group; ANG, angiopoietin; exAH, explants from patients with SAH who underwent transplantation; HC, healthy controls; HCV, non cirrhotic HCV infection group; HCV-C, compensated HCV-related cirrhosis group; NAFLD, non-alcoholic fatty liver disease; ns, not significant; RNA, ribonucleic acid; SAH, severe alcoholic hepatitis.