Detection of novel biomarkers of liver cirrhosis by proteomic analysis

Abstract

Hepatic cirrhosis is a life-threatening disease arising from different chronic liver disorders. One major cause for hepatic cirrhosis is chronic hepatitis C. Chronic hepatitis C is characterized by a highly variable clinical course, with at least 20% developing liver cirrhosis within 40 years. Only liver biopsy allows a reliable evaluation of the course of hepatitis C by grading inflammation and staging fibrosis, and thus serum biomarkers for hepatic fibrosis with high sensitivity and specificity are needed. To identify new candidate biomarkers for hepatic fibrosis, we performed a proteomic approach of microdissected cirrhotic septa and liver parenchyma cells. In cirrhotic septa, we detected an increasing expression of cell structure associated proteins, including actin, prolyl 4-hydroxylase, tropomyosin, calponin, transgelin, and human microfibril-associated protein 4 (MFAP-4). Tropomyosin, calponin, and transgelin reflect a contribution of activated stellate cells/myofibroblasts to chronic liver injury. The expression of tropomyosin, transgelin, and MFAP-4, an extracellular matrix associated protein, were further evaluated by immunohistochemistry. Tropomyosin and MFAP-4 demonstrated high serum levels in patients with hepatic cirrhosis of different causes.

Conclusion: A quantitative analysis of MFAP-4 serum levels in a large number of patients showed MFAP-4 as novel candidate biomarker with high diagnostic accuracy for prediction of nondiseased liver versus cirrhosis [area under receiver operating characteristic curve (AUC) = 0.97, P < 0.0001] as well as stage 0 versus stage 4 fibrosis (AUC = 0.84, P < 0.0001), and stages 0 to 3 versus stage 4 fibrosis (AUC = 0.76, P < 0.0001).

Fig. 1

Proteome pattern of microdissected cirrhotic septa and liver parenchyma cells revealed by the means of 2D-DIGE. For protein analysis, proteins were Cy3 labeled and separated in the first dimension using carrier ampholyte–based isoelectric focusing, and subsequently, in the second dimension, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein patterns were acquired using a confocal fluorescence scanner. Significantly changed proteins are indicated by arrows and respective spot number. (A) 2D gel of cirrhotic septa; (B) 2D gel of liver parenchyma cells from cirrhotic liver; cirrhotic septa. (C) Section of 2D-DIGE image showing reproducible difference of two candidate proteins after microdissection; Tropomyosin (circle) and MFAP-4 (ellipse) were found significantly highly abundant in seven patients suffering from hepatitis C–associated liver cirrhosis using manual microdissection combined with saturation labeling DIGE technique.

Fig. 2

Immunohistochemical stain of tropomyosin, transgelin, and MFAP-4 in normal and cirrhotic liver. (A) Tropomyosin expression in the wall of blood vessels in a portal area of histologically normal liver. Some fibroblasts also show moderate tropomyosin expression (arrows). (B) Tropomyosin expression in the cirrhotic septum (S) of cirrhotic liver. Note the intense staining in the mesenchymal cells and in extracellular matrix. (C) Transgelin in some mesenchymal cells (arrow) in a portal area. (D) Strong expression of transgelin in mesenchymal cells as well as in extracellular matrix of cirrhotic septa (S) in cirrhotic liver. (E) MFAP-4 expression in normal liver. (F) Strong expression of MFAP-4 in cirrhotic cells (original magnification, × 200).

Fig. 3

Abundance of tropomyosin and MFAP-4 in sera of patients with hepatic cirrhosis of different origins. Different sera of patients with HCV-associated liver cirrhosis (1–5), hepatitis B–associated liver cirrhosis (6), and alcohol-induced liver cirrhosis (7–11) in comparison with healthy controls (12–18 for tropomyosin, 12–17 for MFAP-4) were analyzed by means of Western blot. Outer lanes (A 19 and B 18, respectively) show the appropriate positive control.

Fig. 4

Box plot and scatter plot of MFAP-4 sera concentration in patients with hepatic cirrhosis of different origins. The small black circle and the horizontal line mark the mean values and median of the single measurements (triangles) of MFAP-4 serum concentration for HCV-associated and ALD-associated hepatic cirrhosis, respectively; the large boxes constitute 50% of the measurements, whereas the error bars mark the standard deviation.

Fig. 5

Receiver operating characteristics curves based on MFAP-4 sera concentrations in patients with hepatic cirrhosis of different causes. ROC analyses show high diagnostic accuracies of MFAP-4 serum concentration for HCV-associated and ALD-associated hepatic cirrhosis (AUC = 0.97, P < 0.0001 and 0.82, P < 0.0019, respectively).

Fig. 6

Box plot and scatter plot of MFAP-4 sera concentration of patients with different hepatic fibrosis stages on HCV infection. The small black circle and the horizontal line mark the mean values and median of the single measurements (triangles) of the different fibrosis stages (F0–F4), respectively; the large boxes constitute 50% of the measurements, whereas the error bars mark the standard deviation.

Fig. 7

Receiver operating characteristics curves based on MFAP-4 sera concentrations in patients with different fibrosis stages. ROC analyses show high diagnostic accuracies of MFAP-4 serum concentration for early (F0 and F1) and late fibrosis stages (F4).