Plasma levels of matrix metalloproteinase-2 and -9 in male and female patients with cirrhosis of different aetiologies

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Chih-Yang Huang 1 ,2, Kuo-Chih Tseng 3 ,4, Ming-Nan Lin 4 ,5, Jen-Pi Tsai 4 ,6,7, Cheng-Chuan Su 8 ,9

Author Affiliations

1Graduate Institutes of Basic and Chinese Medical Sciences, China Medical University, Taichung, Taiwan

2Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan

3Department of Internal Medicine, Buddhist Dalin Tzu Chi Hospital, Chiayi, Taiwan 4School of Medicine, Tzu Chi University, Hualien, Taiwan

5Department of Family Medicine, Buddhist Dalin Tzu Chi Hospital, Chiayi, Taiwan 6Department of Nephrology, Buddhist Dalin Tzu Chi Hospital, Chiayi, Taiwan 7Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan

8Departments of Clinical Pathology and Anatomic Pathology, Buddhist Dalin Tzu Chi Hospital, Chiayi, Taiwan

9Departments of Laboratory Medicine and Pathology, School of Medicine, Tzu Chi University, Hualien, Taiwan

Correspondence to Dr Cheng-Chuan Su, Department of Clinical Pathology, Buddhist Dalin Tzu Chi Hospital, 2 Minsheng Road, Dalin Town, Chiayi County 622, Taiwan; sucpo@yahoo.com.tw

Received 4 February 2015 Revised 3 June 2015 Accepted 11 June 2015

Published Online First 5 August 2015 Next Section

Abstract

Background Liver fibrosis and cirrhosis may be reversible in some circumstances. Reliable diagnostic tests are necessary for monitoring hepatic fibrogenesis. Matrix metalloproteinase (MMP)-2 and MMP-9 are two of the major MMPs in the

circulation and may be most relevant to hepatic fibrosis. The behaviour of MMPs may be significantly different in men and women and may also differ in cases of cirrhosis of various aetiologies.

Aims To evaluate the manifestations of MMP-2 and MMP-9 in liver cirrhosis of different aetiologies in men and women and to compare these patterns with those of healthy controls.

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patients with cirrhosis and 112 age- and gender-matched healthy controls. We then correlated these MMP levels with gender and disease aetiology.

Results Plasma MMP-2 concentrations in patients showed a trend towards increasing values with cirrhosis severity and were markedly increased in patients regardless of gender and aetiology compared with healthy controls (p<0.0001). Plasma mean MMP-9 levels were comparable in patients with cirrhosis and controls, but increased with disease severity. They were significantly lower in patients (130.5 ng/mL), female patients (85.4 ng/mL) and male patients (150.4 ng/mL) with mild cirrhosis than in controls (163.2 ng/mL), female controls (162.5 ng/mL) and male controls (163.3 ng/mL) (p=0.001, 0.041 and 0.009, respectively). MMP-2 and MMP-9 concentrations were not significantly different between genders among controls and among various patient subgroups.

Conclusions Plasma MMP-2 level may be a useful diagnostic marker for monitoring hepatic fibrogenesis in patients with disease of different aetiologies.

LIVER DISEASE

CHEMICAL PATHOLOGY EXTRACELLULAR MATRIX COLLAGENASE

fibrosis

Previous Section Next Section Introduction

A major histopathological change occurs during the progression of liver disease from hepatitis to cirrhosis, and then to hepatocellular carcinoma (HCC) which involves the amount of extracellular matrix (ECM).1 Liver fibrosis or cirrhosis results from an imbalance between enhanced matrix synthesis by hepatic stellate cells (HSCs) and diminished breakdown of connective tissue proteins by matrix metalloproteinases (MMPs). The net result of this is increased deposition of ECM,2–4 which may be reversible in some circumstances.5–10

Reliable diagnostic tests are necessary for monitoring hepatic fibrogenesis. The extracellular catalytic activity of each MMP is regulated by transcriptional activation at the level of the gene. This activity is also regulated by extracellular cleavage of a latent proenzyme to its active form and is specifically inhibited by the tissue

inhibitors of the metalloproteinase system.4 ,11 Therefore, serum/plasma levels of MMPs may serve as a helpful marker of active fibrosis or cirrhosis.

MMP-2 and MMP-9 are two of the major MMPs in the circulation and are responsible for degradation of the basement membrane, which is composed

primarily of type IV collagen.12 MMP-2 and MMP-9 may be the most relevant MMPs in hepatic fibrosis or cirrhosis. The mechanism of evolution to HCC from cirrhosis

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differs in hepatic disease of different aetiologies. In addition, the prevalence of cirrhosis and HCC differs in men and women.13–15 Hence, the behaviour of MMPs may be different in patients with cirrhosis of various aetiologies and may differ between the genders; however, no comprehensive study of these aspects of cirrhosis has been reported. Our study aimed to evaluate the manifestations of MMP-2 and MMP-9 in liver cirrhosis of different aetiologies in men and women and to compare these patterns with those of healthy controls.

Previous Section Next Section Materials and methods

Healthy controls and study group

After obtaining written informed consent from all subjects and after medical

examinations had been performed, residual plasma samples were collected from 112 patients (21 female and 91 male) with cirrhosis and 112 age- and sex-matched healthy controls. The healthy controls were selected from a group of people receiving routine health examinations during the same period as the study group. Healthy controls were free from hepatitis, cirrhosis and other major diseases. Patients with myocardial infarction, congestive heart failure, diabetes mellitus, malignancy or major trauma were excluded from the study.

Cirrhosis was diagnosed based on the results of liver biopsy tests or on the presence of clinical and laboratory features of portal hypertension, as shown by oesophageal varices at endoscopy and/or collateral circulation on ultrasonography. Disease severity was assessed according to the Child–Pugh scoring system.16

Patients with a history of cirrhosis with positive plasma hepatitis B virus surface antigen (HBsAg) for more than 6 months, negative anti-hepatitis C virus antibody (anti-HCV) and no other apparent cause for chronic liver disease were considered to have hepatitis B virus (HBV)-related cirrhosis. Subjects with positive plasma anti-HCV for more than 6 months, negative HBsAg and no other apparent cause for their chronic liver disease were considered to have HCV-related cirrhosis. Subjects with alcohol-related cirrhosis were defined as those who had consumed at least 80 g of alcohol daily for at least the past 5 years, who were negative for HBsAg and anti-HCV and who had no other apparent cause for their chronic liver disease. Table 1 shows the mean ages and numbers of female and male healthy controls and various subgroups of patients with cirrhosis.

View this table: In this window In a new window Table 1

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subgroups

The study protocol was approved by the institutional review board of the Buddhist Dalin Tzu Chi Hospital, Chiayi, Taiwan.

Sample collection and processing

Each plasma sample was collected in a sterile tube (BD Vacutainer, 3.0 mL, PST gel and lithium heparin 56 U; Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) and centrifuged immediately at 4°C to remove cells. Aliquots of the supernatants were stored at −70°C until needed within 3 months.

Chemiluminescence immunoassay for HBsAg and anti-HCV

Plasma samples were assayed for HBsAg and anti-HCV using the VITROS HBsAg and anti-HCV reagent packs, respectively, with controls and calibrators (Ortho-Clinical Diagnostics, High Wycombe, Buckinghamshire, UK) and VITROS ECi

Immunodiagnostic System (Ortho-Clinical Diagnostics, Rochester, New York, USA). Both were used according to the manufacturer's instructions.

Enzyme immunoassay for MMP-2 and MMP-9

Plasma samples were assayed for MMP-2 and MMP-9 using the MMP-2 and MMP-9 ELISA kits, respectively, with calibrators and negative and positive controls (R & D Systems, Bio-Techne, Minneapolis, Minnesota, USA) and ELISA reader (TECAN Austria GmbH, Salzburg, Austria), according to the manufacturers’ instructions. The MMP-2 assay recognised active MMP-2 and the MMP-9 assay recognised active and pro-MMP-9 enzymes (total pro-MMP-9). The interassay coefficients of variation of replicate samples for MMP-2 were 9.8% at 4.0 ng/mL, 6.9% at 12.4 ng/mL and 5.6% at 19.6 ng/mL. For MMP-9, the interassay coefficients of variation were 7.9% at 1.0 ng/mL, 7.8% at 2.4 ng/mL and 6.9% at 12.2 ng/mL. The ELISA assays were performed in duplicate and the mean values used.

Statistical analysis

Differences between two groups of variables without normal distribution were analysed with the Mann–Whitney test. Statistical significance was set at p<0.05. Statistical analyses were performed using SPSS V.17.0 for Windows (SPSS, Chicago, Illinois, USA).

Previous Section Next Section Results

Female patients in nearly all subgroups were significantly older than the

corresponding male patients (table 1). Patients with HBV-related disease (mean age 49 years; range 27–68 years) were much younger than patients with HCV-related disease (mean age 60.5 years; range 35–80 years) (p=0.0008; Mann–Whitney test). Plasma MMP-2 concentrations were markedly increased in patients with liver cirrhosis, regardless of disease aetiology, compared with MMP-2 concentrations in

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controls (p<0.0001; Mann–Whitney test) and showed a trend towards increasing values with the degree of liver cirrhosis (table 2). MMP-2 concentrations were significantly greater in patients with Child–Pugh class C cirrhosis than in those with class A or B cirrhosis (p<0.0001 and=0.012, respectively; Mann–Whitney test). Plasma mean MMP-9 levels were comparable in patients with cirrhosis and healthy controls, but they increased with disease severity (table 2). Both plasma MMP-2 and MMP-9 concentrations were not significantly different among patients with liver disease of different aetiologies (p>0.05; Mann–Whitney test).

Mean plasma MMP-2 and MMP-9 levels in healthy controls and in patients with cirrhosis

Both plasma MMP-2 and MMP-9 concentrations were not significantly different between both genders among healthy controls and among various subgroups of patients with cirrhosis (p>0.05; Mann–Whitney test).

Generally, the mean plasma MMP-2 levels were significantly greater in the various subgroups of patients, irrespective of gender, than in the healthy controls (tables 3 and 4). MMP-2 levels were significantly greater in female patients with Child–Pugh class C cirrhosis than in female subjects with class A cirrhosis (p=0.027; Mann-Whitney test) and significantly greater in male patients with class C cirrhosis than in men with class A or B cirrhosis (p<0.0001 and p=0.018, respectively; Mann-Whitney test). The mean plasma MMP-9 level was significantly lower in patients, female patients and male patients with Child–Pugh class A cirrhosis than in controls, female controls and male controls (p=0.001, 0.041 and 0.009, respectively; Mann–Whitney test) (table 3).

Comparison of mean plasma MMP-2 and MMP-9 levels between female healthy controls and female patients with cirrhosis

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controls and male patients with cirrhosis Previous Section Next Section

Discussion

Plasma MMP-2 and MMP-9 levels

MMPs have the ability to degrade ECM and, therefore, these enzymes may play an important role in the pathogenesis of liver injury. Under normal physiological conditions, the activities of MMPs are precisely regulated at the level of

transcription, activation of the precursor zymogens, interaction with specific ECM components and inhibition by endogenous inhibitors.11 ,17

Liver fibrosis or cirrhosis is a dynamic process involving an imbalance between matrix synthesis and matrix degeneration, which is characterised by the activation of

HSCs.18 ,19 In the later stages of liver injury and HSC activation, these cells express a combination of MMPs that can degrade normal liver matrix, while inhibiting

degradation of the fibrillar collagens that accumulate in liver fibrosis. This pattern is characterised by the combination of pro-MMP-2 and membrane-type 1 MMP (MT1– MMP) expression, which drives pericellular generation of active MMP-2 (gelatinase A; 72 kDa type IV collagenase) and local degradation of normal liver matrix.19 Northern hybridisation studies have shown a 1.4-fold increase in MMP-2 expression in fibrotic or cirrhotic liver, compared with normal liver.20 As assessed by gelatin-zymography, MMP-2 was detected in human cirrhotic liver but not in normal liver tissue.21 These findings suggest that activated MMP-2 may remodel liver

parenchyma during the process of liver fibrosis or cirrhosis. In accordance with increased production of MMPs in liver tissue samples, serum MMP-2 concentrations were markedly increased in patients with liver cirrhosis compared with controls,22– 25 and showed a good correlation with the degree of liver fibrosis.22 ,25 Our study, which included more cases than earlier studies, also supported these findings. The MMP-2 concentrations were increased in patients with HCV-induced cirrhosis in the studies by Lichtinghagen et al26 and by our group. Nevertheless, in another study, no correlation was found between plasma MMP-2 concentrations and liver fibrosis or cirrhosis in patients with chronic hepatitis C.27

The serum MMP-9 (gelatinase B; 92 kDa type IV collagenase) activities of liver cirrhosis as measured by zymography were reported to be significantly lower than MMP-9 activities among healthy controls.23 Serum MMP-9 levels measured with the ELISA method were also described as lower in patients with cirrhosis than in

controls,25 ,26 and lower with the progression of liver disease.25 In contrast to these findings, plasma levels of MMP-9 measured with ELISA by Hayasaka et al28 were reported to be largely similar in controls and in patients with cirrhosis, just as in our study. However, in our study the plasma MMP-9 levels were lower in patients with

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Child–Pugh class A cirrhosis than in healthy controls and increased with the severity of cirrhosis. Possible explanations for the different results may be differences in study populations, variation in the spectrum of liver disease and the use of different assay systems.

MMP-2 and MMP-9 levels according to aetiology

Patients with alcoholic liver disease have a stronger anti-inflammatory immune status and response and a higher infection rate than patients with virus-induced cirrhosis.29 Accordingly, the manifestations of MMP-2 and MMP-9 in patients with alcohol-induced cirrhosis may be different from those in patients with virus-induced cirrhosis. In a study using the ELISA method, patients with alcohol-related chronic liver disease did not have significantly higher MMP-2 or MMP-9 concentrations than patients with virus-related cirrhosis.25

HBV and HCV are the predominant causes of cirrhosis and HCC and account for most cases of HCC worldwide.30 ,31 Based on genetic alterations, HBV has more direct carcinogenic activity. The severity and inflammatory activity of the cirrhosis are more important in HCV-infected patients than in HBV-infected patients,14 ,32 in whom viral replication is a major predictor of HCC.33 Therefore, the concentrations of MMP-2 and MMP-9 may differ between HBV-infected and HCV-infected patients. In our study, HCV-related patients were much older than HBV-related patients, consistent with published reports that advancing age is a risk factor for HCC among HCV-infected patients.14 However, plasma MMP-2 concentrations were markedly increased in patients with cirrhosis regardless of disease aetiology, compared with controls (p<0.0001). On the other hand, plasma MMP-9 levels in patients with cirrhosis, irrespective of aetiology, did not seem to differ significantly from those in healthy controls. Levels of both plasma MMP-2 and MMP-9 were not significantly different among patients with different aetiologies of disease.

MMP-2 and MMP-9 levels by gender

The prevalence of cirrhosis and HCC is different in men and women.13 ,15 In our previous study, male patients were three times more likely than female patients to be diagnosed with HCC.15 HCC is usually a complication of liver cirrhosis;30 ,34 more than 80% of patients with HCC have cirrhosis.30 Consequently, the activity of MMP-2 and MMP-9 in patients with cirrhosis may be different in men and women. However, the results of this study showed that both plasma MMP-2 and MMP-9 concentrations were not significantly different between men and women among healthy controls and among various subgroups of patients.

In conclusion, plasma MMP-2 levels, and to a lesser extent MMP-9 levels, correlated with the severity of liver disease and may reflect changes in ECM remodelling. The plasma MMP-2 levels were significantly higher among cirrhotic patients, regardless of

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disease aetiology and gender, than among the healthy controls. This suggests that plasma MMP-2 levels may be useful diagnostic markers for monitoring hepatic fibrogenesis in patients with disease of different aetiologies. More studies with larger patient cohorts can identify clinically relevant cut-off values with reliable confidence limits. Our study appears to show that MMP-9 has little value in differentiation between cirrhosis and healthy individuals. The plasma mean MMP-9 levels in cirrhotic patients were generally comparable to those in the healthy controls. They were significantly lower only in patients, female patients and male patients with low-grade cirrhosis, compared with healthy controls, female controls and male controls, respectively. Although the mechanism of evolution to HCC from cirrhosis differs in disease of different aetiologies and the prevalence of cirrhosis and HCC differs in men and women, our study results showed that the plasma levels of MMP-2 and MMP-9 in patients with cirrhosis were not significantly different between men and women and among patients with different disease aetiologies.

Take home messages

Plasma matrix metalloproteinase (MMP)-2 concentrations in patients with cirrhosis showed a trend towards increasing values with cirrhosis severity and were markedly increased in patients regardless of gender and aetiology compared with healthy controls.

Plasma mean MMP-9 levels were comparable in patients with cirrhosis and controls, but increased with disease severity.

Plasma MMP-9 levels were significantly lower in patients, female patients and male patients with mild cirrhosis than in controls, female controls and male controls, respectively.

MMP-2 and MMP-9 concentrations were not significantly different between men and women among controls and among various patient subgroups.

Previous Section Next Section Footnotes

Handling editor Tahir Pillay

Contributors C-YH contributed to the conception, design and important intellectual content of the study and critical revision and final approval of the paper. K-CT, M-NL contributed to acquisition of study data and critical revision and final approval of the paper. J-PT contributed to statistical analysis and critical revision and final approval of the paper. C-CS made a substantial contribution to the conception, design,

organisation, conduct and performance of the study, statistical analysis and writing, critical revision and final approval of the paper.

Funding This study was supported by grants DTCRD96-05 and DTCRD 98-06 from the Buddhist Dalin Tzu Chi Hospital, Chiayi, Taiwan.

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Competing interests None declared.

Ethics approval Institutional review board of the Buddhist Dalin Tzu Chi Hospital, Chiayi, Taiwan.

Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement All relevant data from this study are included in the submission.

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