Serum level of full-length connective tissue growth factor reflects liver fibrosis stage in patients with Fontan-associated liver disease

Abstract

Background: Chronic liver disease leads to liver fibrosis, and an accurate diagnosis of the fibrosis stage is crucial for medical management. Connective tissue growth factor (CTGF) is produced by endothelial cells and platelets and plays a central role in inducing fibrosis in various organs. In the present study, we tested the validity of measuring the serum levels of two types of CTGF to estimate the biopsy-confirmed liver fibrosis stage.

Methods: We used two detection antibodies targeting the N- and C-terminal of CTGF to measure the serum levels of two forms of CTGF consisting of its full length and its N-terminal fragment. We analyzed the level of CTGF (via enzyme-linked immunosorbent assay) and the liver fibrosis stage in 38 patients with Fontan-associated liver disease (FALD) (26 cases of which were diagnosed pathologically). Correlations were determined by multivariate analysis and the area under the receiver operating characteristic curve. The 65 patients with nonalcoholic fatty liver disease (NAFLD) were included as a disease control group for examination.

Results: Full-length CTGF was significantly inversely correlated with liver fibrosis in patients with FALD. Although the platelet count was also associated with the liver fibrosis stage, full-length CTGF was more closely correlated with the fibrosis stage. Furthermore, the level of full-length CTGF was inversely associated with high central venous pressure. Conversely, the serum level of CTGF was not correlated with the fibrosis stage in NAFLD.

Conclusion: The serum level of full-length CTGF may be useful for estimating the liver fibrosis stage in patients with FALD.

Fig 1. CTGF level in patients with FALD with different fibrosis stages.

(A) Two sandwich ELISA assay systems were used. The full-length CTGF level was determined using the antibody against module 1 of CTGF as the capture antibody and the antibody against module 4 of CTGF as the detection antibody. The total CTGF level was determined using the antibody against module 2 of CTGF as the detection antibody, which detected the combined levels of N-terminal CTGF fragment and full-length CTGF. The N-terminal CTGF fragment level was determined by subtracting the full-length CTGF level from the total CTGF level. (B, E) The serum levels of total CTGF (full-length + N-terminal fragment), (C, F) full-length CTGF, and (D, G) N-terminal CTGF fragment were determined in 38 patients with FALD. The liver fibrosis stage was histologically confirmed in 26 patients. Mild fibrosis was assumed to be present in five patients based on their laboratory data and lack of liver cirrhosis characteristics in imaging. (C) The full-length CTGF level was significantly inversely correlated with the liver fibrosis stage (r = –0.68, p < 0.01). There were no statistically significant correlations between the fibrosis stage and the (B) total CTGF level or (D) N-terminal CTGF level. We verified histologically confirmed cases as well. (F) The full-length CTGF level was significantly inversely correlated with the liver fibrosis stage (r = –0.68, p < 0.01), and (G) the N-terminal CTGF level tended to be correlated; however, (E) the total CTGF level was not correlated. All data were determined in duplicate, and the average values are shown. CTGF, connective tissue growth factor; ELISA, enzyme-linked immunosorbent assay; FALD, Fontan-associated liver disease.

Fig 2. Correlation between platelet count and CTGF level in patients with FALD.

(A) The correlation between the fibrosis stage and platelet count was determined in patients with FALD. (B–D) The correlation between the platelet counts and CTGF levels (total, full-length, and N-terminal) was also assessed. (B) The total CTGF level was not associated with the platelet count. (C) The full-length CTGF level was significantly correlated with the platelet count. (D) The N-terminal fragment level tended to be negatively correlated with the platelet count, although the correlation was not statistically significant. All CTGF levels were determined in duplicate, and the average values are shown. CTGF, connective tissue growth factor; FALD, Fontan-associated liver disease.

Fig 3. Correlation between CVP and CTGF level in patients with FALD.

Correlation between CVP and (A) fibrosis stage, (B) platelet count, and (C–E) CTGF levels (total, full-length, and N-terminal). (A) CVP tended to be increased with fibrosis development. The (B) platelet count and (C) total CTGF level were not associated with CVP. (D) The full-length CTGF level tended to be negatively correlated with CVP. (E) The N-terminal fragment level tended to be correlated with CVP, although the correlation was not statistically significant. All CTGF levels were determined in duplicate, and the average values are shown. CTGF, connective tissue growth factor; CVP, central venous pressure; FALD, Fontan-associated liver disease.

Fig 4. CTGF level in patients with NAFLD with different stages of biopsy-proven level fibrosis.

The (A) total CTGF level (full-length + N-terminal fragment), (B) full-length CTGF level, and (C) N-terminal CTGF fragment level in serum from 14 patients with NAFLD with different fibrosis stages were measured. The N-terminal CTGF fragment level was determined by subtraction as described above. All data were obtained in duplicate, and the average values are shown. CTGF, connective tissue growth factor; NAFLD, nonalcoholic fatty liver disease.

Fig 5. Changes in CTGF level at sequential liver biopsy in patients with NAFLD.

Serum levels of (A) total CTGF (full-length + N-terminal fragment), (B) full-length CTGF, and (C) N-terminal CTGF fragment were determined in eight patients with NAFLD who underwent sequential liver biopsy. The blue lines indicate the patients in whom the fibrosis stage pathologically improved. The red lines indicate the patients who did not improve. All data were determined in duplicate, and the average values are shown. CTGF, connective tissue growth factor; NAFLD, nonalcoholic fatty liver disease.