Effect of pegylated interferon alpha 2b plus ribavirin treatment on plasma transforming growth factor-β1, metalloproteinase-1, and tissue metalloproteinase inhibitor-1 in patients with chronic hepatitis C

Abstract

AIM: To evaluate the effect of antiviral treatment on plasma levels of transforming growth factor-β1 (TGF-β1), metalloproteinase 1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in patients with chronic hepatitis C.

METHODS: TGF-β1, MMP-1, and TIMP-1 plasma concentrations were measured by an enzyme immunoassay in 28 patients, during 48 wk of treatment with pegylated interferon-alpha 2b (PEG-IFN-α2b) plus ribavirin (RBV) and after 24 wk of follow-up. Patients were divided into two groups: responders (R) and non-responders (NR) related to achieved sustained virologic response. Normal values were evaluated in plasma samples of 13 healthy volunteers.

RESULTS: Baseline plasma concentrations of TGF-β1 and TIMP-1 (30.9±3.7 and 1 506±61 ng/mL respectively) measured in all subjects significantly exceeded the normal values (TGF-β1: 18.3±1.6 ng/mL and TIMP-1: 1 102±67 ng/mL). In contrast, pretreatment MMP-1 mean level (6.5±0.9 ng/mL) was significantly lower than normal values (11.9±0.9 ng/mL). Response to the treatment was observed in 12 patients (43%). TGF-β1 mean concentration measured during the treatment phase decreased to the control level in both groups. However at wk 72, values of NR patients increased and became significantly higher than in R group. TIMP-1 concentrations in R group decreased during the treatment to the level similar to normal. In NR group, TIMP-1 remained significantly elevated during treatment and follow-up phase and significant difference between both groups was demonstrated at wk 48 and 72. MMP-1 levels were significantly decreased in both groups at baseline. Treatment caused rise of its concentration only in the R group, whereas values in NR group remained on the level similar to baseline. Statistically significant difference between groups was noted at wk 48 and 72.

CONCLUSION: These findings support the usefulness of TGF-β1, TIMP-1, and MMP-1 in the management of chronic hepatitis C. Elevated TIMP-1 and low MMP-1 plasma concentrations during antiviral therapy may indicate medication failure.

Key Words: HCV; Hepatitis; Liver; Interferons; Fibrosis

INTRODUCTION

Transforming growth factor-β1 (TGF-β1) is considered as a pivotal inducer of liver fibrosis acting through activation of hepatic stellate cells (HSCs) and their transformation to myofibroblasts, which are the main source of extracellular matrix (ECM) proteins[1,2]. Moreover, TGF-β1 stimulates the production of tissue inhibitor of TIMP-1 that inhibits MMP activity. This effect is responsible for the inhibition of ECM protein breakdown and its accumulation[3]. TGF-β1 inhibits DNA synthesis serving as a terminator of regenerative cell proliferation and induces apoptosis of hepatocytes[4]. Additionally, TGF-β1 may inhibit stellate cell apoptosis and promote their survival, at least in part as a result of anti-apoptotic effect of TIMP-1[5,6]. On the other hand, TGF-β1 exerts regulatory, mostly immunosuppressive effects on the immune system and as demonstrated recently can also suppress hepatitis C virus (HCV) replication[7,8]. Since HCV infection is related to an immune response, cell proliferation and fibrosis as well as modulation of TGF-β1 can affect the course of chronic hepatitis C. As demonstrated recently, HCV core and nonstructural proteins regulate biological functions in HSC and increase the secretion of TGF-β1 and the expression of ECM proteins in both HSCs and parenchymal hepatic cells[9,10]. The possible role of TGF-β1, TIMP-1, and MMP-1 as predictive biomarkers of chronic hepatitis activity and progression is supported by recent clinical studies[11-19]. These studies demonstrated association with hepatic function impairment or fibrosis, and only few evaluated possible effects of antiviral treatment on growth factors, but they did not include possible metalloproteinase involvement[20,21].

We undertook this study to evaluate the effect of pegylated interferon-α2b plus ribavirin (PEG-IFN-α2b/RBV) treatment on plasma TGF-β1, TIMP-1, and MMP-1 levels in patients with chronic hepatitis C.

MATERIALS AND METHODS

Patients

Ethical approval for the study was obtained from the Bioethical Committee of the Medical University of Bialystok. Informed consent was obtained from 28 patients (8 females and 20 males, mean age 49±12 years) with chronic hepatitis C, who were included into the protocol of PEG-IFN-α2b (Pegintron™, Schering-Plough) and RBV (Rebetol™, Schering-Plough) treatment. All patients had proven chronic hepatitis C through the presence of anti-HCV antibodies with elevated ALT activities demonstrated at least twice during a 6-mo observation period. Additionally, the disease activity was confirmed by the presence of viral replication and liver biopsy (Hepafix System, Braun, Melsungen, Germany). Patients with HBV infection and a history of alcohol abuse or psychiatric disorders were excluded from the study. Patients received combination therapy with weekly doses of 100 μg PEG-IFN-α2b administered subcutaneously and RBV administered orally at daily doses of 1 000 or 1 200 mg/d based on body weight <75 or ≥75 kg, respectively. The total duration of treatment was 48 wk. Liver biopsy was performed before and after antiviral therapy. Patients were divided into two groups related to sustained virologic response (SVR), defined as undetectable HCV RNA, 24 wk after the end of therapy. Patients who achieved SVR were included into the responder group (R) and those without SVR into non-responder group (NR). Paraffin-embedded biopsy specimens were stained and evaluated using the scoring system according to Scheuer[22]. TGF-β1, TIMP-1, and MMP-1 plasma concentrations were measured at baseline, 24 and 48 wk after treatment and additionally 24 wk after the termination of the treatment (wk 72). Serum liver function tests and scored histological changes were investigated for the possible correlation with TGF-β1, TIMP-1, and MMP-1. Normal values of TGF-β1, TIMP-1, and MMP-1 were collected from 13 healthy volunteers (5 females and 7 males , mean age: 48±6 years).

Methods

Venous blood for plasma TGF-β1, TIMP-1, and MMP-1 was collected on ice using tubes with EDTA. Samples for TGF-β1 were immediately activated with acetic acid and urea and assayed with ELISA using recombinant human TGF-β soluble receptor Type II (TbR-II) as a solid phase precoated onto a microplate (Quantikine^®, R&D Systems Inc., Minneapolis, USA) as described previously[23]. TIMP-1 and MMP-1 were assayed by the two-site ELISA sandwich technique (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) using specific antibodies as a solid phase. MMP-1 assay recognized total human MMP-1, namely free and complexed with TIMP-1. TIMP-1 assay recognized total human TIMP-1, including free and complexed with any of the metalloproteinases bound to the solid phase. TIMP-1 or MMP-1 bound to the solid phase was detected by peroxidase-labeled antibodies. There was no cross-reactivity between TIMP-1 and MMP-1 in these assays. Alanine and aspartate aminotransferase (ALT and AST) activity and bilirubin concentration were measured in serum using a Cobas Mira instrument (Roche).

Statistical analysis

Values were expressed as mean±SE. The significance of the difference was calculated by two-tailed Student's t test. For correlation analysis, the Pearson’s product moment correlation was performed. P<0.05 was considered statistically significant.

RESULTS

Plasma concentrations of TGF-β1 and TIMP-1 measured before PEG-IFN-α2b/RBV treatment (mean: 30.9±3.7 and 1 506±61 ng/mL respectively) significantly exceeded the normal values (18.3±1.6 and 1 102±67 ng/mL respectively). Treatment resulted in a significant decrease of TGF-β1 by wk 24, and its further decline at the end of the treatment as well as 24 wk after its completion to the level similar to normal (Table 1). TIMP-1 plasma mean concentration also decreased, but did not differ significantly from baseline. Moreover, it remained on the level significantly exceeding controls during treatment and follow-up period (Table 1). Mean MMP-1 baseline level (6.5±0.9 ng/mL) was significantly lower than normal (11.9±0.9 ng/mL) but increased during the treatment. After treatment, its level still remained lower than normal but the difference was not significant (Table 1). There was a significant positive correlation between TIMP-1 and aminotransferases as well as between TGF-β1 and AST at baseline (Table 2). A significant correlation was also demonstrated between baseline TGF-β1 or TIMP-1 concentrations and scored fibrosis in pre-treatment liver biopsy specimens (Table 3). No association was demonstrated between MMP-1 and biochemical or histological signs of liver injury (Tables 2 and 3).

Table 1 Plasma concentrations of TGF-β1, TIMP-1, and MMP-1 during treatment (mean±SE).

	Controls	Weeks after starting treatment
		0	24	48	72
TGF-β1 (ng/mL)	18.3±1.6	30.9±3.7a	21.2±2.8c	17.9±2.0c	21.0±2.5c
TIMP-1 (ng/mL)	1 102±67	1 506±61a	1 372±70a	1 389±51a	1410±66a
MMP-1 (ng/mL)	11.9±0.9	6.5±0.9a	7.2±1.5a	7.5±2.6	8.0±1.6

^aP<0.05 vs normal,

^cP<0.05 vs baseline.

Table 2 Correlation expressed by r-value between biochemical indices of liver injury and TGF-β1, TIMP-1, or MMP-1 in chronic hepatitis C patients before treatment.

	Bilirubin	ALT	AST
TGF-β1 (ng/mL)	0.24	0.163	0.388a
TIMP-1 (ng/mL)	0.023	0.393a	0.370a
MMP-1 (ng/mL)	–0.192	–0.130	–0.299

^aP<0.05 biochemical indices vs TGF-β₁, TIMP-1, and MMP-1.

Figure 2 Mean TGF-β1 (A), TIMP-1 (B), and MMP-1 (C) plasma concentrations before and during IFN-α plus RBV therapy as well as 24 wk after its completion (wk 72) in respect to the treatment efficacy. Statistical significance in comparison to normal values is indicated with asterisks and between groups with arrows.

Table 3 Correlation expressed by r-value between scored histological picture and TGF-β1, TIMP-1, or MMP-1 in chronic hepatitis C patients before treatmen.

	Inflammation		Fibrosis
	Portal	Lobular
TGF-β1 (ng/mL)	0.088	–0.132	0.495a
TIMP-1 (ng/mL)	0.091	0.326	0.404a
MMP-1 (ng/mL)	0.229	0.36	–0.018

^aP<0.05 histological score vs TGF-β₁, TIMP-1, and MMP-1

SVR was observed in 12 among 28 patients (43%). Evaluation of baseline liver function tests showed no statistically significant differences between R and NR groups (Table 4). Treatment did not affect bilirubin levels in both groups. Responders demonstrated a significant decrease of ALT and AST activities during the treatment and follow-up. Decline of aminotransferases activity in NR group was only temporal and rose to values significantly higher than in R group after discontinuation of the treatment (Table 4). As demonstrated in Figure 1, scored values of histologic changes were similar before the treatment. There were no significant differences between biopsies preformed before and after the treatment in NR group. In contrast, responders demonstrated improvement after the treatment; however, statistically significant difference between score values at wk 0 and 48 was noted only in respect to portal inflammation.

Figure 1 Mean score values of histologic changes in liver biopsy specimens before (wk 0) and after (wk 48) treatment. Statistically significant difference is indicated with arrows.

Table 4 Values of biochemical indices of liver injury during treatment (0, 24, and 48 wk) and 24 wk after its completion (wk 72) in both groups.

	Weeks after the beginning of treatment
	0	24	48	72
Bilirubin	Non-responders	1±0.1	1±0.1	0.7±0.1	0.9±0.1
(mg%)	Responders	1.1±0.1	1.1±0.1	0.9±0.1	1.0±0.1
ALT(U/L)	Non-responders	101±15	40±6c	55±20	98±18a
	Responders	96±13	35±9c	24±5c	22±2c
AST(U/L)	Non-responders	58±8	30±4c	34±6ac	64±10a
	Responders	48±6	26±3c	20±1c	19±2c

^aP<0.05 responders vs non-responders,

^cP<0.05 vs baseline.

There were no statistically significant differences in TGF-β1, TIMP-1, and MMP-1 concentrations between R and NR groups at the baseline and 24 wk after the treatment. As demonstrated in Figure 2A, TGF-β1 mean concentration decreased to the control level during treatment in both groups. However, 24 wk after the treatment (wk 72), values in NR patients increased (23.2±2.3 ng/mL) and became significantly higher than those in R group (18.6±3.7 ng/mL). As shown in Figure 2B, mean concentration of TIMP-1 decreased during the treatment only in R group and there were no statistically significant differences in comparison with controls at wk 24, 48, and 72. In contrast, TIMP-1 concentration in NR group remained significantly elevated (above 1 500 ng/mL) during treatment and follow-up (Figure 2B). Significant difference between both groups was demonstrated at wk 48 and 72. As shown in Figure 2C, MMP-1 levels were significantly decreased in both groups at baseline. Treatment caused rise of its concentration only in the R group, whereas the values in NR group remained on the level similar to baseline. Statistically significant difference between groups was noted at wk 48 and 72 (Figure 2C).

DISCUSSION

The effect of TGF-β1 on liver fibrosis is at least in part related to stimulation of TIMP-1 that affects MMP activity and is responsible for inhibition of ECM protein breakdown[3]. The pivotal role of TGF-β1 in fibrogenesis is initially proved in transgenic mice with overexpression of TGF-β1, causing increase of its plasma levels up to 700 ng/mL and a marked upregulation of TIMP-1 gene expression[24-26]. Recent studies demonstrated that HCV proteins can stimulate secretion of TGF-β1 and production of ECM proteins by HSCs[9,10]. On the other hand, Murata et al[8] showed that TGF-β suppresses viral HCV-RNA replication and can affect the mechanism of liver disease caused by HCV. Chronic liver injury leading to fibrosis displays diminished ECM degradation mainly through TIMP induction following MMP inhibition[3]. As demonstrated recently, TIMP-1 recombinant plasmid has inhibitory effects on the production of types I and III collagens secreted by activated rHSCs in vitro[27].

The most important factor affecting TGF-β1 measurement in human beings is from platelets which are an important source of this cytokine[28]. The Quantikine ELISA System is recommended because of quick and simple activation with acid and urea that disrupt the majority of TGF-β1 complexes. Mean plasma concentration of TGF-β1 measured in our healthy controls with this method is consistent with the range from more than 20 studies reviewed by Grainger et al[28].

According to our previous research, TGF-β1 and TIMP-1 correlate with the degree of liver insufficiency, hepatocyte injury and degree of fibrosis in human beings with liver cirrhosis and chronic viral hepatitis[12,23,29]. Association between TGF-β1 mRNA in liver specimens and fibrogenic activity in chronic hepatitis is demonstrated for the first time by Castilla et al[30]. Ten years after the association between circulating or tissue TGF-β and liver fibrosis in HCV infection has been confirmed by Kanzler et al[13]. As demonstrated by Yoo et al[31] and Lee et al[32], HBV antigens also stimulate TGF-β1 synthesis. According to Neuman et al[20,33], serum TNF-α reflects the progression of inflammation, whereas TGF-β reflects the degree of fibrosis in HCV patients. A similar relationship has been demonstrated with respect to primary biliary cirrhosis and alcoholic liver disease[34]. Our previous study showed that a positive predictive value of TGF-β1 plasma levels exceeding the upper normal range reaches 96% for liver cirrhosis[23]. According to Boeker et al[11] measurement of plasma TIMP-1 detects cirrhosis with 100% sensitivity but a lower specificity. Lichtinghagen et al[14] demonstrated that MMP-1 mRNA expression increases steadily with fibrosis progression during the course of chronic hepatitis C. Walsh et al[17] who studied liver histology in patients with chronic hepatitis C have underlined the high sensitivity of TIMP-1 and TIMP-2 in detecting advanced liver disease. According to Nie et al[35], there is a significant correlation between circulating and liver levels of TIMP-1 in cirrhotics, indicating that its measurement in plasma may be useful in fibrosis management. These observations indicate the usefulness of both TGF-β1 and TIMP-1 as possible early non-invasive biomarkers for liver fibrosis.

In this study, we confirmed the association between the degree of hepatocyte injury or liver fibrosis and plasma TGF-β1 or TIMP-1 levels in patients with chronic hepatitis C. As the levels of TGF-β1 showed a similar behavior in both groups during therapy, it is unclear whether its decrease is a direct effect of medication on the expression or an effect caused by HCV inhibition. However, measurement carried out 24 wk after treatment demonstrated an association with treatment efficiency. Similar effects on plasma TGF-β1 have been observed by Castilla et al[30] and Neuman et al[20] and in our previous study of chronic hepatitis B[29]. TIMP-1 and MMP-1 concentrations demonstrated significant differences between groups at the end of the treatment and after 24 wk of follow-up. Since plasma TIMP-1 and MMP-1 remained on the baseline level in non-responder group only, lack of their normalization should be considered as a possible indicator of ineffective antiviral therapy. Results of the present study are in accordance with our previous findings, demonstrating the strong association between TGF-β1 or TIMP-1 plasma levels and scored hepatic fibrosis evaluated in biopsy specimens of patients with chronic hepatitis B and C[12]. Since the findings of increased TGF-β1 and TIMP-1 are accompanied with an elevation in plasma carboxyterminal cross-linked telopeptide of type 1 procollagen (ICTP), indicating type I collagen degradation, collagenolytic mechanisms precede TGF-β1/TIMP-1 dependent stimulation of liver fibrosis[12]. Low MMP-1 plasma levels before the treatment in the present study are consistent with this observation as well as in accordance with Murawaki et al[15] who demonstrated a decrease in MMP-1 concentration during histological progression of chronic hepatitis. Moreover, significantly decreased baseline plasma MMP-1 followed by an increase during treatment supports the role of TGF-β1/TIMP-1 dependent mechanism of liver fibrosis in patients with active chronic hepatitis C. Similar effects on MMP-1 and TIMP-1 in patients with chronic hepatitis C have been observed by Ninomiya et al[16] who showed improvement of liver histology after treatment with IFN-α alone. Downregulation of the mechanism causing an increase of MMP-1 activity should be considered as the probable reason for this effect. As we demonstrated recently, treatment of chronic hepatitis B with lamivudine affects TGF-β1, TIMP-1, and MMP-1 plasma levels in a similar way and this mechanism should be recognized as an effect of response to the antiviral treatment, irrespective of the etiology[29].

Results of this study support the role of TIMP-1 and MMP-1 balance in the TGF-β1 dependent mechanism of liver fibrosis related to HCV infection. Association between hepatic injury and antiviral treatment efficacy suggests their possible usefulness in chronic hepatitis C management. Elevated TIMP-1 and low MMP-1 plasma concentrations during antiviral therapy may indicate medication failure.