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Connective tissue growth factor in serum as a new candidate test for assessment of hepatic fibrosis.

Exaggerated production of extracellular matrix is a life-threatening complication of chronic inflammatory liver injury of various etiologies. The process leading to excess deposition of collagens, proteoglycans, and glycoproteins in liver parenchyma (fibrosis) is an important feature of liver cirrhosis, the end stage of chronic liver diseases characterized by complete scarring of the organ. Thus, detection and follow-up of fibrogenesis is mandatory for early treatment and risk stratification; but, until now, there have been no reliable and cost-effective non-invasive measures available (1).

On the basis of the molecular pathogenesis of fibrosis, we initiated a diagnostic evaluation of connective tissue growth factor (CTGF; gene symbol CTGF) in serum. CTGF is a 36-38 kd cysteine-rich, heparin-binding protein that belongs, with Cyr61 and NOV, to the CCN family of proteins regulated by transforming growth factor [beta] (TGF-[beta]) and is also designated CCN2 (2). Its role in fibrogenesis suggests this protein as a potential fibrogenic marker, because (i) CTGF is a downstream mediator of TGF-[beta], which is the most important fibrogenic cytokine (3); (ii) CTGF is expressed increasingly in the major profibrogenic cell type, i.e., hepatic stellate cells, during their transdifferentiation in extracellular matrix-producing myofibroblasts (4, 5); (iii) expression of CTGF is strongly upregulated in fibrotic liver tissue (4, 5); and (iv) CTGF is secreted into the extracellular space and, thus, can reach the systemic circulation directly. Because CTGF was reported to be associated with and released by platelets during the coagulation process (6, 7), and because thrombocytopenia is associated with advanced fibrosis and cirrhosis, we also investigated the relationship of platelet count and CTGF.

CTGF was measured in sera of patients with chronic liver diseases (n = 83; 48 men, 35 women) who were admitted to our hospital. We used histologic examination of liver biopsies to establish diagnoses of liver fibrosis without histologic signs of cirrhosis (16 patients) and fully developed cirrhosis [50 patients of alcoholic (n = 23), chronic viral hepatitis C (n = 10) and hepatitis B (n = 4), and cryptogenic (n = 13) etiology]. Results of carbohydrate-deficient transferrin in serum did not indicate actual alcohol abuse. We also included an additional group of patients with serologically proven chronic viral hepatitis B (n = 3), C (n = 10), and both (n = 4) who did not have a biopsy, but were suspected to have developed fibrosis. An allocation of patients of this group to either fibrosis or cirrhosis could not be made. The control population (n = 74) consisted of blood donors and healthy volunteers.

Sera were stored at -70 [degrees]C for 3 to 4 months before analysis. Assay plates were coated with rabbit anti-CTGF (H-55, Santa Cruz) diluted in PBS-coating buffer pH 7.4 for 24 h at room temperature with gentle shaking followed by blocking unspecific binding sites with blocking solution [10 g/L bovine serum albumin, 50 g/L sucrose, 5 g/L sodium azide in PBS pH 7.4]. A reagent diluent of 50 [micro]L [10 g/L bovine serum albumin in PBS; pH 7.4] was added to each well, followed by 50 [micro]L of calibrators (BioVendor) or serum samples in duplicate to the microwells and incubated overnight at 4 [degrees]C with shaking. After 4 washings with PBST [PBS including 0.5 ml/L Tween 20], goat anti-CTGF (L-20, Santa Cruz) antibody was added and incubated for 2 h with shaking. Specificity of anti--CTGF-antibodies for CTGF was proven by Western blot analysis of serum proteins showing the typical-38-kd band and a half full-length molecule (18-19 kd). The plate was washed, followed by incubation with biotinylated rabbit antigoat IgG (DakoCytomation) for 1 h. After 4 washings and incubation with streptavidin-HRP (Dako), the plates were rinsed and substrate reagent TMB (R&D Systems) was added to each well. Color development was stopped after 20 min at 37 [degrees]C, and absorbance was measured at 450 nm. The calibration curves were fitted through use of a 4-parameter logistic function provided by the VICTOR Multilabel Counter (WALLAC).

[FIGURE 1 OMITTED]

The inter- and intraassay imprecisions (CVs) were 8.7% and 12% (134 [micro]G/L), respectively. The detection limit was 20 ~g/L. We routinely counted platelets in EDTA-blood samples of patients and probands using the CD-4000 hematologicanalyzer (Abbott).

CTGF serum concentrations as well as CTGF/platelet count ratios were summarized by minimum, maximum, median, mean, and quantiles (lower quartile, median, upper quartile), separately for the 4 different patient/ proband subgroups. The nonparametric Wilcoxon ranksum test was used for pair-wise comparison of groups. The global significance level was chosen as a = 5%. No [alpha]-adjustment for multiple testing was carried out. The software packages R, version 2.1.0 (Free Software Foundation), and Medcalc (Mariakere) were used.

CTGF-concentrations were higher (P <0.001) in sera of patients with chronic hepatitis, established fibrotic disease, and cirrhotic liver diseases than in healthy controls (Fig. 1). The mean concentration was highest in the fibrosis (5.2-fold above control) and chronic hepatitis group (4.3-fold). CTGF was lower (P = 0.0034) in cirrhotic patients than in fibrosis patients. For other pair-wise comparisons of patient subgroups CTGF concentrations did not differ (P >0.05). A reduction of platelet count (by ~50%) was noticed only in the cirrhosis group, and no correlation of platelet count with the CTGF concentration was seen in any group (r = 0.0456). The ratio of CTGF concentration to the platelet count (Table 1) was higher (P <0.001) than in the control group in all groups with chronic liver diseases and highest in the cirrhotic patient group, which had the lowest mean platelet counts (Fig. 1). Ratios of the cirrhosis and fibrosis groups were not different, nor were the ratios of the fibrosis and hepatitis group (P >0.05). The areas under the ROC curves of CTGF (CTGF/platelet ratio) for fibrosis vs control and cirrhosis vs control were 0.955 (0.926) and 0.887 (0.946), respectively. The corresponding 95% confidence intervals were (0.890-0.987) (0.850-0.971) and (0.818-0.937) (0.889-0.978). The CTGF/platelet ratio increased the detection limit for cirrhosis from 84% to 92% and the specificity from 85% to 87.5% at cutoffs for CTGF of 364 [micro]G/L and ratio of 2.05.

The data confirm our hypothesis that CTGF increases in the circulation of patients with active, fibrogenic liver diseases and, hence, suggest CTGF as a potential new noninvasive marker of ongoing fibrogenesis. Future cross-sectional and prospective studies are needed of CTGF concentrations in acute hepatic diseases (e.g., acute hepatitis) to evaluate parenchymal cell necrosis as a potential mechanism of increased CTGF in the circulation, because this protein is also present in hepatocytes (unpublished). Our data support a preliminary report on biliary atresia, which indicated circumstantially increased CTGF concentrations in progressive liver disease (8). Increase of CTGF concentrations in plasma, serum (9,10), and urine (11) have been suggested as surrogate markers of fibrotic disease activity in scleroderma (9,10), pulmonary fibrosis (10), diabetic nephropathy (11,12), and glomerulosclerosis of other causes (11). However, no systematic studies of serum CTGF in fibrotic liver diseases are currently available. The significantly lower concentration of CTGF in cirrhotic patients compared with fibrosis patients might reflect decreased fibrogenic activity in this end stage of fibrotic disease. Fibrogenic activity in fully developed cirrhosis is slowed down; therefore, CTGF concentrations in the circulation may not reflect the extent of a scarring (fibrosis) but rather than an ongoing (active) fibrogenesis (i.e., the dynamic process of fibrosis development). From the clinical point of view, the latter information is much more relevant, because it opens the possibility for therapeutic intervention and monitoring of antifibrogenic trials. To evaluate the possibility that CTGF concentrations reflect active fibrogenesis indeed requires studies of patients with chronic hepatitis who have varying degrees (grades) of activity of disease but similar stages of fibrosis. This pilot study warrants further studies of CTGF as an early fibrogenic marker protein in the circulation.

References

(1.) Afdhal NH, Nunes D. Evaluation of liver fibrosis: a concise review. Am J Gastroenterol 2004;99:1160-74.

(2.) Rachfal AW, Brigstock DR. Connective tissue growth factor (CTGF/CCN2) in hepatic fibrosis. Hepatol Res 2003;26:1-9.

(3.) Gressner AM, Weiskirchen R, Breitkopf K, Dooley S. Roles of TGF-/3 in hepatic fibrosis. Front Biosci 2002;7:d793-807.

(4.) Williams EJ, Gapa MDA, Brigstock DR, Arthur MJP, Benyon RC. Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells. J Hepatol 2000;32:754-61.

(5.) Paradis V, Dargere D, Vidaud M, de Gouville A-C, Huet S, Martinez V, et al. Expression of connective tissue growth factor in experimental rat and human liver fibrosis. Hepatology 1999;30:968-76.

(6.) Cicha I, Garlichs CD, Daniel WG, Goppelt-Struebe M. Activated human platelets release connective tissue growth factor. Thromb Haemost 2004; 91:755-60.

(7.) Kubota S, Kawata K, Yanagita T, Doi H, Kitoh T, Takigawa M. Abundant retention and release of connective tissue growth factor (CTGF/CCN2) by platelets. J Biochem 2004;136:279-82.

(8.) Tamatani T, Kobayashi H, Tezuka K, Sakamoto S, Suzuki K, Nakanishi T, et al. Establishment of the enryme-linked immunosorbent assay for connective tissue growth factor (CTGF) and its detection in the sera of biliary atresia. Biochem Biophys Res Commun 1998;251:748-52.

(9.) Dziadzio M, Usinger W, Leask A, Abraham D, Black CM, Denton C, et al. N-terminal connective tissue growth factor is a marker of the fibrotic phenotype in scleroderma. J Med 2005;98:485-92.

(10.) Sato S, Nagaoka T, Hasegawa M, Tamatani T, Nakanishi T, Takigawa M, et al. Serum levels of connective tissue growth factor are elevated in patients with systemic sclerosis: association with extent of skin sclerosis and severity of pulmonary fbrosis. J Rheumatol 2000;27:149-54.

(11.) Riser BL, Cortes P, DeNichilo M, Deshmukh PV, Chahal PS, Mohammed AK, et al. Urinary CCN2 (CTGF) as a possible predictor of diabetic nephropathy: preliminary report. Kidney International 2003;64:451-8.

(12.) Roestenberg P, van Nieuwenhoven FA, Wieten L, Boer P, Diekman T, Wiersinga WM, et al. Connective tissue growth factor is increased in plasma of type 1 diabetic patients with nephropathy. Diabetic Care 2004;27:116470.

D01: 10.1373/cfinchem.2006.070466

M. Gressner, [1 *] Eray Yagmur, [1] Birgit Lahme, [1] Olav Gressner, [3] and Sven Stanzel [2] (1 Institute of Clinical Chemistry and Pathobiochemistry, Central Laboratory, [2] Institute of Medical Statistics, RWTH University Hospital, Aachen, Germany; [3] Department of Internal Medicine I, University Hospital Bonn, Bonn, Germany; * address correspondence to this author at: Institute of Clinical Chemistry and Pathobiochemistry-Central Laboratory, RWTH-University Hospital, Pauwelsstr. 30, 52074 Aachen, Germany; fax 49-0-241-8082512; e-mail gressner~rwth-aachen.de)
Table 1. Descriptive summary of serum CTGF concentrations and
CTGF/platelet count ratios in patients with chronic liver diseases.

 Lower
Patient/proband group N Minimum quartile, 25% Median

CTGF-Serum concentration,
[micro]g/L

 Control 74 26.5 119.5 198.8
 Chronic hepatitis (C) 17 512.0 800.5 974.5
 Fibrosis (A) 16 526.0 1264.0 1805.0
 Cirrhosis (B) 50 150.5 503.5 894.8

CTGF/platelet count ratio

 Control 72 0.09532 0.4588 0.7905
 Chronic hepatitis (C) 17 1.825 2.798 4.754
 Fibrosis (A) 16 0.7781 5.051 6.622
 Cirrhosis (B) 50 1.079 3.963 7.766

Platelet count is
calculated as g/L.

 Mean Upper
Patient/proband group value quartile, 75% Maximum

CTGF-Serum concentration,
[micro]g/L

 Control 399.7 293.9 4954.0
 Chronic hepatitis (C) 1727.0 2388.0 5645.0
 Fibrosis (A) 2088.0 2778.0 5016.0
 Cirrhosis (B) 1273.0 1607.0 5894.0

CTGF/platelet count ratio

 Control 1.494 1.68 16.69
 Chronic hepatitis (C) 7.603 10.31 25.89
 Fibrosis (A) 9.336 11.08 38.73
 Cirrhosis (B) 11.730 13.53 63.38

Platelet count is
calculated as g/L.
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Title Annotation:Technical Briefs
Author:Gressner, Axel M.; Yagmur, Eray; Lahme, Birgit; Gressner, Olav; Stanzel, Sven
Publication:Clinical Chemistry
Date:Sep 1, 2006
Words:1968
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