Tenascin expression in human chronic liver disease and in hepatocellular carcinoma

Abstract: Tenascin is an oligomeric glycoprotein of the extracellular matrix synthesized during embryonic development. It is prominently expressed in a variety of tumors. The role of tenascin in liver tissue is, however, unknown. We used immunocytochemistry to define the localization of tenascin and compare this with the localization of non-collagenous proteins, such as laminin and fibronectin, in normal human liver and pathological liver from patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. In normal liver, tenascin expression was localized along the sinusoidal and vascular wall. In fibrotic liver, tenascin was also observed in the region between the hepatic parenchyma and the fibrosing portal tracts, especially in areas of piecemeal necrosis in chronic hepatitis. Immuno-EM study of liver tissue in chronic hepatitis strongly suggested the synthesis and secretion of tenascin by fat-storing cells into the space of Disse. In hepatocellular carcinoma, tenascin was expressed in both the capsule and lobular septa, but not in the sinusoidal walls of the tumors. These results led us to postulate a close relationship between the occurrence of this protein and disease processes such as fibrosis and cancer invasion.     

Expression of the novel extracellular matrix component tenascin in normal and diseased human liver. An immunohistochemical study.

The novel extracellular matrix glycoprotein tenascin was studied immunohistochemically in normal and fibrotic human liver. Its localization was compared to that of laminin, fibronectin and collagen type IV. In the normal liver, a weak staining for tenascin was detected along sinusoids, while portal tracts were negative. In both alcoholic and cholestatic liver disease and acute and chronic hepatitis, sinusoidal immunoreactivity for tenascin was variably increased as compared to the normal liver. Most striking, however, was the preferential accumulation of tenascin at connective tissue-parenchymal interfaces between proliferating ductules and in areas of piecemeal necrosis. As compared to laminin, fibronectin and collagen type IV, tenascin has the most restricted distribution. Our findings indicate that tenascin is a component of the extracellular matrix of the human liver. Its preferential expression at connective tissue-parenchymal interfaces in fibrosing areas in contrast to its absence from mature fibrous septa suggest a transient role in early matrix organization.     

Serum tenascin levels in chronic liver disease

To evaluate the diagnostic significance of tenascin, the extracellular matrix glycoprotein in chronic liver disease, serum tenascin levels were measured by a newly developed ELISA in 21 patients with chronic persistent hepatitis, in 55 with chronic active hepatitis, in 59 with liver cirrhosis, in 31 with hepatocellular carcinoma, in 26 with acute hepatitis and in 66 healthy subjects. The serum tenascin level was significantly elevated in the patients with chronic active hepatitis, liver cirrhosis, hepatocellular carcinoma, and acute hepatitis when compared with the healthy subjects (P<0.001). The serum tenascin level also increased with increasing severity of chronic liver diseases. A significant correlation was observed between the serum tenascin levels and serum levels of various extracellular matrix proteins such as type III procollagen N-aminoterminal peptide (PIIIP), laminin and the 7S domain of type IV collagen (P<0.001). A strong positive correlation was observed between the serum tenascin levels and histologic findings, particularly in the degree of hepatic fibrosis. This is the first report documenting serum tenascin level increases in patients with various chronic liver diseases. The measurement of the serum tenascin levels may provide additional information relevant to the study of connective tissue.     

Vascular adhesion molecules in acute and chronic liver inflammation.

Adhesion to and penetration through the sinusoidal vascular endothelium is a mandatory step for leukocyte migration and accumulation at sites of liver inflammation. This leukocyte trafficking is controlled by interactions between adhesion molecules on leukocytes and corresponding ligands on endothelial cells. We have analyzed the in situ distribution of two recently described vascular adhesion molecules (i.e., endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1) and of the lymphocyte "homing" receptor cluster of differentiation antigen-44 in normal and inflamed liver biopsy specimens. Endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1 were absent from normal liver tissue, but they were strongly expressed on sinusoidal lining cells in inflammatory liver disease. Endothelial leukocyte adhesion molecule-1 expression predominated diffusely throughout the liver parenchyma in acute hepatitis; in contrast, vascular cell adhesion molecule-1 was mainly expressed in areas of periportal and intralobular inflammation in chronic active and persistent hepatitis. The "homing" receptor cluster of differentiation antigen-44 was weakly expressed on scattered mononuclear cells and on sinusoidal lining cells in normal liver tissue, but it was strongly up-regulated on mononuclear inflammatory cells and sinusoidal lining cells in acute and chronic hepatitis. In addition, reactivity for the cluster of differentiation antigen-44 was found on the membranes of variously sized clusters of hepatocytes in biopsy specimens with acute hepatitis. De novo or up-regulated expression of these adhesion molecules on sinusoidal lining cells in inflamed liver biopsy specimens indicates that these cells actively modulate their phenotype in response to environmental factors, thus playing a key role in the recruitment of leukocytes in acute and chronic liver inflammation.     

细胞间粘附分子－1在乙型肝炎中的表达及意义

为观察细胞间粘附分子－１（ＩＣＡＭ－１）在乙型肝炎肝组织内的表达状况，探讨ＩＣＡＭ－１在乙型肝炎肝细胞免疫损伤中的作用。采用链菌素亲生物蛋白法（ＬＳＡＢ），以ＩＣＡＭ－１的单克隆抗体，检测１０４例急、慢性乙型肝炎，９例无症状携带者，１０例正常肝组织标本内ＩＣＡＭ－１抗原表达情况。结果发现：ＩＣＡＭ－１在正常肝细胞无表达，肝窦内皮细胞弱表达；ＨＢＶ感染后肝细胞呈不同程度的表达，肝窦内皮表达增强；中、重度慢性肝炎，活动性肝硬化肝细胞ＩＣＡＭ－１表达较急性肝炎，轻度慢性肝炎显著增强（Ｐ＜０．０１）；急性肝炎较轻度慢性肝炎显著增强（Ｐ＜０．０１）；轻度慢性肝炎较无症状携带者，正常肝组织显著增强（Ｐ＜０．０１）。表明ＩＣＡＭ－１在乙型肝炎肝细胞内表达与肝细胞损伤有关，对ＨＢＶ的清除可能起着重要作用，ＩＣＡＭ－１在肝窦内皮的表达有助于淋巴细胞向肝组织内浸润。     

Hepatic regeneration in humans with various liver disease as assessed by Ki-67 staining of formalin-fixed paraffin-embedded liver tissue

Abstract: The ability to document nuclear proliferation in the liver is essential to our understanding of hepatic regeneration and hepatocellular carcinoma. While numerous tests are available to provide such information in experimental animals, few can be applied to patients with liver disease. Ki-67 is a proliferating nuclear antigen present in replicating cells. Recent data indicates that staining for Ki-67 reflects nuclear proliferation in these tissues. To date, the technique has had only limited application to human liver tissues in formalin-fixed paraffin embedded tissue. In the present study, we documented Ki-67 staining in archival liver tissue from patients with mild chronic hepatitis, severe chronic hepatitis, inactive cirrhosis, hepatocellular carcinoma, and in normal livers. We found that Ki-67 staining was increased in patients with mild chronic hepatitis (labelling index 29±25), severe chronic hepatitis (labelling index 41 ±40), and hepatocellular carcinoma (labelling index 71 ±61), when compared to patients with inactive cirrhosis, (labelling index 1.4±3.1), and normal livers (labelling index 2.5±3.2). In conclusion, Ki-67 maybe a useful tool to assess hepatocyte proliferation in formalin-fixed paraffin-embedded human liver tissue.     

Intrahepatic accumulation of nitrotyrosine in chronic viral hepatitis is associated with histological severity of liver disease

BACKGROUND/AIMS: The toxicity of nitric oxide is thought to be engendered, at least in part, by its reaction with superoxide yielding peroxynitrite, a potent oxidant that promotes the formation of nitrotyrosine within cells and tissue lesions. In this study we assessed the intrahepatic localization and distribution of the inducible nitric oxide synthase (iNOS) and nitrotyrosine (NTY) in patients with viral and non-viral liver disease. METHODS: We carried out single and double immunostaining experiments on cryostat liver biopsy sections using monoclonal antibodies against iNOS and NTY. We also performed a comparative analysis between the intrahepatic immunostaining score of NTY and the histological activity index of chronic viral hepatitis. RESULTS: We found a marked hepatocellular expression of iNOS with a diffuse lobular pattern in all liver samples from patients with viral liver disease, whereas NTY localization was mainly restricted to cellular foci consisting of hepatocytes and Kupffer cells. Interestingly, we demonstrated by means of double immunostaining experiments the existence of hepatocellular co-localization of iNOS and NTY in the majority of NTY-expressing liver cells. The amount of NTY was significantly higher in liver biopsies from viral liver disease than in non-viral liver disease. In addition, a statistically significant association between the intrahepatic amount of NTY and the severity of viral liver disease was found. CONCLUSIONS: Nitric oxide-mediated nitration of hepatocellular proteins is markedly induced in the inflamed liver tissue from patients with chronic viral hepatitis, and appears to be associated with the histological severity of viral chronic liver disease.     

Hepatitis B virus antigens in liver tissue in hepatocellular carcinoma and advanced chronic liver disease-relationship to liver cell dysplasia

Hepatitis B surface (HBs) and core (HBc) antigens (Ag) were studied in liver tissue in HBsAg seropositive patients with chronic liver disease complicated (n = 32) and not complicated (n = 36) by hepatocellular carcinoma. Both groups were matched by age, sex and underlying disease. There was no qualitative and quantitative difference in tissue HBsAg between the two groups. However, HBcAg was significantly less in quantity in hepatocytes of patients with hepatocellular carcinoma compared to chronic liver disease without cancer. Serum hepatitis B e antigen tested by radioimmunoassay was also less frequently positive in patients with hepatocellular carcinoma. These findings seem to suggest that hepatitis B virus replication becomes less active in the process of hepatocarcinogenesis. The relationship between intrahepatic hepatitis B antigens and liver cell dysplasia was also studied. In hepatocellular carcinoma, tissue hepatitis B antigens often coexisted in the same liver having liver cell dysplasia, but no such association was observed in chronic liver disease without cancer. However, no indication was obtained that the dysplastic cells harbor HBsAg more frequently than non-dysplastic cells.     

Hepatitis B virus antigens in liver tissue in hepatocellular carcinoma and advanced chronic liver disease,relationship to liver cell dysplasia

ABSTRACT— Hepatitis B surface (HBs) and core (HBc) antigens (Ag) were studied in liver tissue in HBsAg seropositive patients with chronic liver disease complicated (n=32) and not complicated (n=36) by hepatocellular carcinoma. Both groups were matched by age, sex and underlying disease. There was no qualitative and quantitative difference in tissue HBsAg between the two groups. However, HBcAg was significantly less in quantity in hepatocytes of patients with hepatocellular carcinoma compared to chronic liver disease without cancer. Serum hepatitis B e antigen tested by radioimmunoassay was also less frequently positive in patients with hepatocellular carcinoma. These findings seem to suggest that hepatitis B virus replication becomes less active in the process of hepatocarcinogenesis. The relationship between intrahepatic hepatitis B antigens and liver cell dysplasia was also studied. In hepatocellular carcinoma, tissue hepatitis B antigens often coexisted in the same liver having liver cell dysplasia, but no such association was observed in chronic liver disease without cancer. However, no indication was obtained that the dysplastic cells harbor HBsAg more frequently than non-dysplastic cells.     

Serum concentration of E-selectin in patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma

Objectives: High levels of soluble E-selectin have been reported in acute and chronic inflammatory disorders. Moreover, in some types of tumor elevated values have been found while in other types reduced levels have been reported. Our aims were to determine whether soluble E-selectin levels might be useful in monitoring the progression of chronic liver disease, including hepatocellular carcinoma. Methods: Circulating soluble E-selectin was measured by an enzyme-linked immunosorbent assay in the sera of 18 patients with chronic hepatitis, 44 with liver cirrhosis, and 38 with hepatocellular-carcinoma-associated liver cirrhosis. Immunohistochemical localization of E-selectin was also performed on liver tissue specimens of patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Results: Serum levels of soluble E-selectin were higher in the chronic hepatitis and liver cirrhosis patients than in the hepatocellular carcinoma patients and healthy controls. Levels in the hepatocellular carcinoma patients and controls were not significantly different. In the liver cirrhosis group, divided according to the Child-Pugh classification, soluble E-selectin decreased with disease severity. Similarly, in patients with liver cirrhosis who developed hepatocellular carcinoma, soluble E-selectin decreased as the disease progressed. Immunohistochemical localization showed strong membrane staining on endothelial cells in areas rich in inflammatory cells in severe chronic hepatitis. In some hepatocellular carcinoma tissues a marked E-selectin staining was observed on endothelial cells of tumor-associated small vessels. Conclusions: The results obtained suggest that high serum levels of soluble E-selectin are associated with chronic hepatitis and liver cirrhosis, and that levels decrease in liver cirrhosis patients as the disease progresses. Patients with hepatocellular carcinoma have different types of soluble E-selectin behaviour the significance of which requires further investigation. Received: 27 July 1999 / Accepted: 24 December 1999     

Histochemical properties of vascular and sinusoidal endothelial cells in liver diseases

Liver biopsy specimens with or without liver diseases were examined immunohistochemically to determine the distribution of endothelial cell markers, factor VIII-related antigen (FVIII-RAg), Ulex europaeus agglutinin I (UEA-I) lectin and PAL-E. We also investigated the localization of laminin, a component of the basement membrane. In normal livers, FVIII-RAg, UEA-I and laminin were negative in sinusoidal endothelial cells, but positive in blood vascular endothelia of the portal area. The antigen detected by PAL-E was distributed in venous endothelial cells. PAL-E did not label endothelial cells of the artery. In the lobule, immunoreactivity with PAL-E was weakly detected only in some sinusoids of the periportal area. In chronic active hepatitis and liver cirrhosis, FVIII-RAg and UEA-I stained endothelial cells of neovasculatures in the enlarged portal areas of the fibrous septum surrounding pseudolobules. Some sinusoidal endothelial cells in cirrhotic livers were reactive to UEA-I and FVIII-RAg, whereas PAL-E-positive cells were found rarely in the pseudolobules. In carcinomatous sinusoidal endothelial cells, FVIII-RAg, UEA-I and PAL-E were strongly stained. Laminin underlay these carcinomatous sinusoids. These suggest capillarization of sinusoids in hepatocellular carcinoma. The histochemical approach using endothelial cell markers could be a practical tool in the diagnosis of hepatocellular carcinoma.     

Immunohistochemical study on tissue inhibitors of metalloproteinases in normal and pathological human livers

The localization of tissue inhibitor of metalloproteinases (TIMP) in normal and pathological livers was examined by immunohistochemistry using monoclonal antibodies at the light microscopic level. In normal liver, immunoreactive TIMP was detected in smooth muscle cells and endothelial cells of blood vessels, fibroblasts, bile duct cells and Kupffer cells, indicating that TIMP is likely to be a general element of the liver. Immunoreactivity was observed in newly-formed blood vessels, proliferating bile ductules, and fibroblasts in the expanded portal area and fibrous septa of chronic active hepatitis and cirrhosis. TIMP was strongly stained in the capsule of hepatocellular carcinoma. The intensity of the immunoreaction in the capsule was generally greater than that in cirrhotic liver apart from the tumor mass. In three of five cases with hepatocellular carcinoma, endothelial walls in contact with tumor cells were positive.     

Histochemical properties of vascular and sinusoidal endothelial cells in liver diseases.

Liver biopsy specimens with or without liver diseases were examined immunohistochemically to determine the distribution of endothelial cell markers, factor VIII-related antigen (FVIII-RAg). Ulex europaeus agglutinin I (UEA-I) lectin and PAL-E. We also investigated the localization of laminin, a component of the basement membrane. In normal livers, FVIII-RAg, UEA-I and laminin were negative in sinusoidal endothelial cells, but positive in blood vascular endothelia of the portal area. The antigen detected by PAL-E was distributed in venous endothelial cells. PAL-E did not label endothelial cells of the artery. In the lobule, immunoreactivity with PAL-E was weakly detected only in some sinusoids of the periportal area. In chronic active hepatitis and liver cirrhosis, FVIII-RAg and UEA-I stained endothelial cells of neovasculatures in the enlarged portal areas of the fibrous septum surrounding pseudolobules. Some sinusoidal endothelial cells in cirrhotic livers were reactive to UEA-I and FVIII-RAg, whereas PAL-E-positive cells were found rarely in the pseudolobules. In carcinomatous sinusoidal endothelial cells, FVIII-RAg, UEA-I and PAL-E were strongly stained. Laminin underlay these carcinomatous sinusoids. These suggest capillarization of sinusoids in hepatocellular carcinoma. The histochemical approach using endothelial cell markers could be a practical tool in the diagnosis of hepatocellular carcinoma.     

Expression of vascular endothelial-cadherin in human hepatocellular carcinoma tissues

Aim: Angiogenesis is important in tumor growth and progression to metastasis. Vascular endothelial (VE)-cadherin is an endothelial cell-specific cadherin required for angiogenesis, but its expression in hepatocellular carcinoma (HCC) tissues has not been examined. Methods: Expression of VE-cadherin was analyzed in 31 HCC frozen tissue specimens by immunohistochemical and immunoelectron microscopic procedures. In addition, the association of its expression with clinicopathological parameters was investigated to determine the possible diagnostic or predictive value of VE-cadherin expression in neoplastic and non-neoplastic liver lesions. Results: Immunoreactive VE-cadherin expression was faint or barely detectable on sinusoidal endothelial cells of normal liver but was evident on sinusoidal or capillary endothelium of chronic hepatitis, cirrhosis, and HCC tissues. VE-cadherin expression was more intense on capillary endothelium of HCC tissues in 26 (84%) of 31 patients than on sinusoidal endothelium of surrounding non-tumorous liver tissues with chronic liver diseases. The intensity or intracapillary extent of positive stain for VE-cadherin on capillary endothelium of HCC tissues was significantly associated with tumor size, capsular invasion and tumor cell differentiation in HCC. Conclusions: Intense VE-cadherin expression was evident in capillary endothelium of HCC tissues, giving the first indication of association with clinicopathological features of HCC patients.     

Immunohistochemical study on tissue inhibitors of metalloproteinases in normal and pathological human livers.

The localization of tissue inhibitor of metalloproteinases (TIMP) in normal and pathological livers was examined by immunohistochemistry using monoclonal antibodies at the light microscopic level. In normal liver, immunoreactive TIMP was detected in smooth muscle cells and endothelial cells of blood vessels, fibroblasts, bile duct cells and Kupffer cells, indicating that TIMP is likely to be a general element of the liver. Immunoreactivity was observed in newly-formed blood vessels, proliferating bile ductules, and fibroblasts in the expanded portal area and fibrous septa of chronic active hepatitis and cirrhosis. TIMP was strongly stained in the capsule of hepatocellular carcinoma. The intensity of the immunoreaction in the capsule was generally greater than that in cirrhotic liver apart from the tumor mass. In three of five cases with hepatocellular carcinoma, endothelial walls in contact with tumor cells were positive.     

Enhanced expression of endothelial nitric oxide synthase and caveolin-1 in human cirrhosis

BACKGROUND/AIMS: Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases (NOS). Caveolin, the principal structural protein in caveolae, interacts with endothelial NOS (eNOS) leading to enzyme inhibition by a reversible process modulated by Ca++ -calmodulin. The aim of the present study was to examine the localizations of eNOS and caveolin-1 at protein level in normal human liver tissue, and how the expressions are altered in cirrhotic liver. METHODS: Fresh liver specimens were obtained from hepatic surgeries. Normal portions resected from cases of carcinoma metastasized to the liver were used as control specimens, and cirrhotic portions resected from cases of hepatocellular carcinoma with hepatitis C-related cirrhosis were used as cirrhotic specimens. Anti-eNOS and anticaveolin-1 antibodies were used for immunohistochemistry and Western blotting. Immunoelectron microscopy was conducted on ultra thin sections using immunoglobulin-gold combined with silver staining. RESULTS: Immunohistochemistry revealed that both eNOS and caveolin-1 were sparsely expressed on hepatic sinusoidal lining in normal liver specimens, and these findings were confirmed by Western blot. Both immunohistochemistry and Western blotting demonstrated over-expression of eNOS and caveolin-1 in cirrhotic liver specimens. Morphometric analysis of immunogold particle labeling for eNOS and caveolin-1 was performed on immunoelectron micrographs. In normal liver tissue, hepatic stellate cells and sinusoidal endothelial cells (SEC) expressed low levels of caveolin-1, and SEC expressed a very low level of eNOS. In cirrhotic liver, both caveolin-1 and eNOS expressions were significantly increased by approximately four-fold on SEC compared to normal liver. CONCLUSION: In cirrhotic human liver, marked increase of caveolin-1 in perisinusoidal cells may promote caveolin-eNOS binding and reduce the activity of eNOS despite an increased eNOS expression, leading to impaired NO production and increased hepatic microvascular tone.     

Early lesions and development of primary hepatocellular carcinoma in man--association with hepatitis B viral infection.

It is well known that primary hepatocellular carcinoma could be derived from chronic hepatitis and liver cirrhosis in epidemiologic studies. However, it is still not clear what kinds of hepatocyte are premalignant cells. Recently we have focused on liver cell dysplasia as a possible premalignant cell, and showed localization of alpha-fetoprotein in the cytoplasma of these cells. Although the dysplastic cells were often seen in the liver of chronic active hepatitis, hepatitis B virus associated DNA polymerase activity was also significantly high in the sera from the patients with chronic active hepatitis. In this paper, we discuss the possible role of hepatitis B virus through hepatocarcinogenesis in human.     

Differential gene expression between chronic hepatitis B and C hepatic lesion

BACKGROUND & AIMS: Complementary DNA (cDNA) microarray technology allows simultaneous expression analysis of hundreds to thousands of genes. We applied the cDNA microarray technique to clarify gene expression profiles in chronic viral hepatitis tissue lesions. METHODS: We made cDNA microarrays consisting of 1080 human cDNAs and analyzed gene expression using labeled cDNAs prepared from 6 normal, 12 chronic hepatitis B, and 14 chronic hepatitis C liver tissues. Relative expression ratios of individual genes were obtained by comparing hybridization of Cy5-labeled cDNAs from chronic hepatitis lesions and Cy3-labeled cDNA from normal liver tissue. RESULTS: Hierarchical clustering analysis of the gene expression profiles in 26 patients showed that the patients were clustered into 2 groups with respect to similarities in differentially expressed genes. Hepatitis B and C virus infection, but not age, sex, or histology of hepatitis, were significant factors determining clustering (P < 0.05). In hepatitis B tissue lesions, genes involved in inflammation were predominant, whereas in hepatitis C, expression of anti-inflammatory response genes was relatively dominant. CONCLUSIONS: These findings shed new light on the possible differential molecular mechanisms in the pathogenesis of hepatitis caused by hepatitis B virus and hepatitis C virus infection, from which hepatocellular carcinoma frequently develops.     

Pathological observations of sinusoid lining endothelial cells and the basement membrane in human hepatocellular carcinoma

OBJECTIVE : To observe changes in sinusoid lining endothelial cells (SEC), type IV collagen (CoIV) and laminin (LM) in chronic liver disease and hepatocellular carcinoma (HCC). To assess the clinicopathological significance of these changes in HCC. METHODS : Thirty specimens were taken from 30 cases of HCC (together with corresponding non‐cancerous tissues), 10 cases of liver cirrhosis, five cases of mild chronic hepatitis and and four cases of normal liver tissues. The specimens were tested for CD34, CoIV and LM by using immunohistochemical methods. CD34, CoIV and LM were semiquantitatively analyzed and assessed in the context of the clinical and pathological features of HCC. RESULTS : CD34 and LM were not present along the sinusoidal walls in normal human liver, however, CoIV was weakly and discontinuously distributed along the sinusoidal walls. In cirrhosis, positive expression of CoIV increased significantly in the sinusoidal walls and became continuous and homogeneous. CD34 and LM were weakly present in the perinodules in a few cases of cirrhosis with obvious inflammatory infiltration. Hepatocellular carcinoma showed a diffuse capillarization, with overexpression of CD34, CoIV and LM. CoIV and LM expression were reduced in poorly differentiated HCC and HCC with portal vein thrombosis. This was frequently accompanied by breaks and losses in the basement membrane. The expression of CD34 in tumors of < 2 cm in diameter was significantly lower than that in tumors of > 5 cm in diameter. The expression of CD34 and LM was markedly increased in HCC compared with non‐cancerous liver tissues. CONCLUSIONS : Diffuse capillarization with overexpression of CD34, CoIV and LM are features of HCC. Frequent breaks in, loss of and decrease of the basement membrane in poorly differentiated tumors and tumors with portal vein infiltration suggests potential metastasis of tumor cells and may play a major role in the metastasis of HCC. CD34 is a useful marker for distinguishing HCC from non‐cancerous liver tissues.