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Morphometric analysis and immunohistochemical expression of cyclooxygenase-2 in hepatitis C virus-induced fibrosis

Abbas, Naglaa F.; Badawi, Manal A.; Abd El-Aal, Wafaa E.; El-Sharkawy, Sonia L.

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doi: 10.1097/01.MJX.0000437951.43008.8d
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Hepatitis C virus (HCV) infection is a significant cause of morbidity and mortality, infecting more than 170 million people worldwide 1. Egypt reports the highest prevalence of HCV worldwide, ranging from 6% to more than 40%, with an average of 13.8% 2. In response to the viral infection, the patients develop liver fibrosis and most of the related morbidity occurs after the development of cirrhosis with the risk of hepatocellular carcinoma (HCC) 3.

Multiple signaling pathways are activated in response to the viral infection, which helps in the regulation of the gene expression related to inflammation 4. Cyclooxygenase (COX) is the first step in the biosynthesis of various inflammatory mediators including prostaglandins and thromboxanes. Two COX isoforms COX-1 and COX-2 have been found to be the key enzymes that control the rate-limiting step in the conversion of arachidonic acid to prostaglandins 3. COX-1 is constitutively expressed in a number of cell types and is involved in the homeostatic functions of prostaglandins 5. COX-2 is induced by a variety of proinflammatory stimuli such as cytokines and lipopolysaccharides and has been implicated in inflammation, fibrogenesis and carcinogenesis 6,7. COX-2 is also upregulated in the liver under conditions of chronic viral hepatitis or cirrhosis 8,9. Moreover, growing evidence indicates that COX-2 plays an important role in several signaling pathways in hepatic stellate cells, and its expression is further upregulated by cytokines 10.

The HCV core protein was able to upregulate COX-2 expression in hepatocyte-derived cells, providing a potential mechanism for hepatic fibrosis during chronic HCV infection 4,11,12.

Assessment of liver fibrosis is a very important diagnostic and prognostic evaluation in chronic liver diseases including HCV-induced liver fibrosis 13. To clarify the role of COX-2 in the progression of liver fibrosis, several studies have been investigated in animal models to show its role in the exacerbation and amelioration of fibrogenesis after treatment with COX-2 inhibitors; however, its definitive role has not been elucidated fully 14,15.

The first widely applied scoring system for inflammation and fibrosis in chronic hepatitis was that of Knodell et al.16. Because this system failed to provide a sufficiently clear separation of fibrosis and inflammatory activity, Ishak et al.17 proposed a modification of the Knodell system that attempted to address this and other deficiencies. It consisted of a seven-tier staging system for fibrosis, with values ranging from 0 (no fibrosis) to 6 (cirrhosis).

‘Morphometry’ was defined as the quantitative description of structure. This structure may be macroscopic or microscopic in size. The reasons for the increasing interest in the application of morphometry in diagnostic pathology are its advantages in terms of objectivity, reproducibility and the possibility of detecting minor differences or variations in a specimen that would otherwise escape subjective evaluation 18.

Quantitative analysis of histologic images attempts to enhance diagnostic pathology in three ways: by isolating the application of individual criteria, thus contributing to their objective use; by increasing precision in the evaluation of quantitative criteria; and by evaluating features that are simply not appreciated by the human eye 19.

All histological scoring systems of liver fibrosis incorporate a categorical description of architectural changes, without reference to quantitative changes in the liver collagen (fibrosis), as the stage of liver disease progresses or regresses 20. Quantitative measurement of the severity of liver fibrosis was achieved using computerized image analysis in Masson trichrome-stained sections. The results showed a highly significant increase in the area of fibrosis in the case of rats treated with carbon tetrachloride 21.

This study aimed to assess HCV-induced liver fibrosis using morphometric image analysis and to study the relation between the extent of fibrosis and COX-2 expression as one of the important enzymes involved in fibrogenesis.

Patients and methods

Liver biopsies of 42 cases were the subject of the current study: 27 men and 15 women with a mean age of 43.3±12.5 years, obtained from diverse sources.

Histological staging according to fibrosis

The Ishak system was used to evaluate the stage of hepatic fibrosis, consisting of seven descriptive criteria from no fibrosis to cirrhosis with corresponding scores assigned from 0 to 6 Ishak score 17. In this study, the cases were categorized into low-fibrosis stages (Ishak score 1–3, n=21) and high-fibrosis stages (Ishak score 4–6, n=21).

The fibrosis area% for each liver biopsy specimen was calculated using a Leica-Qwin 500 Image Analyzer (damaged area software) (LEICA Imaging Systems Ltd, Cambridge, England) in the Pathology Department, National Research Center.

Cyclooxygenase-2 immunohistochemical staining

COX-2 expression was investigated in all tissues using the streptavidin–biotin technique. Four-μm-thick sections were deparaffinized, hydrated and incubated in 3% hydrogen peroxide for 30 min to block the internal peroxidase activity. Antigen retrieval was performed by microwave pretreatment for 10 min in 0.01 mol/l citrate buffer. Slides were incubated at 4°C overnight with anti-COX-2 monoclonal antibody at a dilution of 1 : 50 (Lab Vision Corporation) (Fremont, California USA). These steps were followed by a 30-min incubation with biotinylated horse anti-mouse antibody at room temperature, and then avidin–biotin–peroxidase complex for 60 min at room temperature and finally diamiobenzidine for 3–5 min. The slides were counterstained with hematoxylin, dehydrated and mounted. The negative control was obtained by omitting the primary antibody.

The cyclooxygenase-2 immunohistochemical score

The COX-2 immunohistochemical staining score was assessed using the scoring system by Pazirandeh et al.9, which is based on a sum of two parameters: the staining intensity and the staining distribution. The COX-2 staining intensity was scored using a scale of 0–3 (0=no staining; 1=weak staining; 2=moderate staining; 3=strong staining).

The distribution of COX-2 staining was scored using a scale of 0–2 (0=no staining; 1=focal/patchy staining; 2=diffuse staining).

Each specimen was evaluated for the sum of these two parameters and assessed by the degree of COX-2 immunoreactivity using a quantitative score that ranged from 0 to 5. Scores between 0 and 3 were categorized as low COX-2 expression and scores of 4 and 5 were categorized as high COX-2 expression.

Statistical analysis

Statistical analysis was performed using the statistical software program SPSS version 9.02 (SPSS Inc., Chicago, Illinois, USA). Qualitative data were expressed by number and percentage, whereas quantitative data were expressed as mean±SD. Comparison between qualitative data was carried out by the χ2-test, whereas the Student t-test was used for qualitative data to compare the means of two groups. Significant values were at P less than 0.05.


Histological staging of liver fibrosis according to the Ishak scoring system

This study included 42 cases of chronic hepatitis. Fibrosis scores were assigned for each biopsy specimen according to the criteria of Ishak et al.17. Eight cases were of stage 1 (19%) (Fig. 1a and b), nine were of stage 2 (21.4%), four were of stage 3 (9.5%), 10 were of stage 4 (23.8%), five were of stage 5 (11.9%), and six were of stage 6 (14.2%) (Fig. 2a and b). Accordingly, 21 cases were of high-fibrosis stage and 21 were of low fibrosis.

Fig. 1:
(a) Chronic hepatitis stage 1/6 Ishak scoring system (H&E, ×100). The portal area showed expansion by fibrous tissue. (b) Binary image of the fibrous tissue in the portal area by the image analysis system.
Fig. 2:
(a) Chronic hepatitis stage 6/6 Ishak scoring system (H&E, ×100). Marked bridging fibrosis, wide fibrous septa and cirrhotic nodule formation. (b) Binary image of the wide fibrous tissue by the image analysis system.

The relation between the fibrosis stage according to the Ishak system and the morphometric fibrosis area% of fibrosis is shown in Table 1, revealing a significant relation between them (P=0.022), where the mean morphometric fibrosis area increased with increasing stage of fibrosis according to Ishak.

Table 1:
Relation between the Ishak scoring system and the morphometric fibrosis area%

Immunohistochemical expression of cyclooxygenase-2

Hepatocyte cytoplasmic staining for COX-2 was noted in all cases of hepatitis either with low or high fibrosis. According to Pazirandeh et al.9, five cases were of score 2, 11 cases were of score 3, 16 cases were of score 4, and 10 cases were of score 5. Accordingly, 16 of 42 cases (38.1%) were of low COX-2 expression and the remaining 26 cases (61.9%) were of high COX-2 expression (Figs 3 and 4).

Fig. 3:
Low cyclooxygenase-2 expression in a case of chronic hepatitis with low-stage fibrosis (imunohistochemistry, ×100).
Fig. 4:
Chronic hepatitis with high-stage fibrosis showing high cyclooxygenase-2 expression (imunohistochemistry, ×100).

Cyclooxygenase-2 expression and severity of hepatic fibrosis

COX-2 expression scores based on the degree of hepatic fibrosis according to the Ishak staging using a full range of scores (1–6) revealed a nonsignificant relation (P>0.05) (Table 2). However, high COX-2 expression was detected in 85.7% of the cases with a high Ishak staging, but only in 38.1% of cases with a low Ishak stage revealing a significant relation between COX-2 expression and the degree of hepatic fibrosis (P=0.007) (Table 3). There was an insignificant relation between COX-2 expression and the morphometric fibrosis area% (P>0.05) (Table 4).

Table 2:
Relation between cyclooxygenase-2 expression and the full range of Ishak scoring system
Table 3:
Relation between cyclooxygenase-2 expression and the Ishak scoring system
Table 4:
Relation between cyclooxygenase-2 expression and the morphometric fibrosis area%


Liver fibrosis is characterized by an abnormally excessive accumulation of extracellular matrix resulting from chronic inflammation of the hepatic parenchyma. HCV is one of the most common causes of liver fibrosis and cirrhosis with the major risk of development of HCC 22. COX-2 has been implicated in tissue injury and fibrogenesis in animal models, but little is known regarding its role in HCV-related liver disease in humans 3. However, previous studies showed that hepatocellular COX-2 expression occurred by the synergistic inducing effect exerted by HCV proteins and several inflammatory mediators that are involved in the fibrotic process 23.

In this study, 42 cases of HCV-induced liver fibrosis were assessed using morphometric image analysis, and the relation between the extent of fibrosis and COX-2 expression was studied. In the present study, 21 cases (50%) were of high-fibrosis stage and 50% were of low-fibrosis stage according to the Ishak staging.

The use of quantitative morphometric measurements of the severity of hepatic fibrosis may help more efficiently than other quantitative descriptions for the assessment of fibrosis 13. The Ishak staging showed a significant positive relation with the mean fibrosis area% using image analyzer for morphormetric measurement of fibrosis. This was consistent with a previous study of Friedenberg et al.24 who evaluated 91 patients with chronic hepatitis and compared the fibrosis ratios calculated by an image analysis program and Ishak scores, providing a significant quantitative correlation.

Several reports in the literature have addressed the measurement of liver fibrosis by computerized image analysis to derive a ratio of fibrosis to the total area of the biopsy specimen and compared it with variable ordinal histopathological scoring systems. The present study showed an overall statistically significant positive relation between the fibrosis ratio and the ordinal score, in agreement with other studies using the Ishak scoring system. O’Brien et al. 25 evaluated 230 patients with hepatitis C and compared the fibrosis ratios calculated by an image analysis program and Ishak scores. Hui et al.26 validated a new image analysis system, Bioquant Nova Prime, in estimating the collagen content in liver biopsy samples from patients with chronic hepatitis B. The biopsies were stained with picrosirius red, and the areas of collagen were measured. The results were correlated with laboratory parameters and Ishak-modified histological scores. Finally, Friedenberg et al.27 evaluated 91 patients with chronic hepatitis and compared the fibrosis ratios calculated by an image analysis (FibroXact) program and Ishak scores. All the studies reported a statistically significant correlation between fibrosis assessed by image analysis and the respective fibrosis classifications used. Goodman 28 quantified liver fibrosis progression by a computer-assisted morphometric image analysis in sections stained with picrosirius red, evaluating patients with treatment-refractory chronic hepatitis C enrolled in a placebo-controlled clinical trial of interferon γ1b for the treatment of advanced hepatic fibrosis. Each patient had paired biopsies performed before and after treatment. Morphometry was found to be a more sensitive tool than histological staging to demonstrate fibrosis progression.

In the current study, we have shown that all studied cases of hepatitis with low or high fibrosis expressed COX-2 protein. However, 61.9% of the cases exhibited high COX-2 expression and 38.1% exhibited low expression according to Pazirandeh et al.9. This was consistent with previous results of El-Bassionny et al.29, who showed that intrahepatic expression of COX-2 was increased in HCV patients with increased inflammatory activity and fibrotic stage, suggesting its possible pathogenic role in the process of fibrogenesis. Other previous studies of Nunez et al.3 and Mohammed etal.31 also showed that COX-2 overexpression is associated with progressive hepatic fibrosis in HCV infection and that the HCV core was able to upregulate COX-2 gene expression in liver cells, providing a potential mechanism for hepatic fibrosis during HCV infection. Mohammed et al.31 further demonstrated that COX-2 overexpression in liver cirrhosis is possibly due to the overproduction of prostaglandins, the major component of the inflammation and hyperdynamic circulation associated with cirrhosis.

On correlating COX-2 expression scores based on the degree of hepatic fibrosis according to the Ishak staging using the full range of scores (0–6), our study revealed a nonsignificant relation. However, when the Ishak staging score was assessed as high and low stages, the current study revealed significant correlation between COX-2 expression and the degree of Ishak fibrosis. This was consistent with the study of Jeong et al.30 who followed Pazirandeh et al.9 and Qiu et al.32 for the staining score of COX-2 expression. However, they suggested that the role of COX-2 in liver fibrosis is not fully understood, although several investigators have reported data from animal models demonstrating the efficacy of COX-2 inhibitor in liver fibrosis.

Mohammed et al.31 reported that as COX-2 is believed to contribute to tumor development, high COX-2 expression could be a contributor to HCC development in cirrhosis. They suggested that COX-2 upregulation in cirrhosis could provide a possible new role for selective COX-2 inhibitors in reducing inflammation and minimizing the occurrence of HCC in patients with cirrhosis. In addition, Tu et al.33 reported that selective inhibition of COX-2 by rofecoxib in vivo reduced portal hypertension and was associated with antifibrotic activity occurring in HCV hepatitis. However, Yu et al.15 demonstrated that COX-2 inhibitors cause many side effects including potential renal, gastrointestinal and cardiovascular (mainly myocardial infarction) events.

In conclusion, morphometric assessment of liver fibrosis using image analysis has been proven to be a simple and accurate method for evaluating hepatic fibrosis. Moreover, COX-2 expression is significantly correlated with the extent of fibrosis assessed by morphometry, suggesting its role in the progression of hepatic fibrosis. Further investigation including an animal study to assess the safety of selective therapy by COX-2 inhibitors and their effects on hepatic fibrosis may be required.

No title available.


Conflicts of interest

There are no conflicts of interest.


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