Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver.

BACKGROUND/AIMS: Angiogenesis is essential in liver regeneration. However, only little is known about sinusoidal endothelial cell proliferation and the role of different angiogenic growth factors and their receptors during regeneration. METHODS: Seventy percent hepatectomy was carried out on male rats. Serial changes in endothelial cell proliferation were evaluated by immunohistochemistry. The mRNA expressions of angiogenic growth factors (vascular endothelial growth factor (VEGF) and angiopoietins 1 and 2) and their receptors (flt-1, flk-1, Tie-1 and Tie-2) in the whole liver were evaluated by semi-quantitative RT-PCR. RESULTS: Significant elevation of endothelial cell proliferation started at 48 h and peaked at 72 h after hepatectomy. The ratio of sinusoids to liver tissue area initially decreased at 72 h, and thereafter, significantly increased at 96 h. VEGF related factors had early peaks, which coincided with the endothelial proliferation. flt-1, flk-1 and VEGF expressions peaked at 24, 48 and 72 h, respectively. angiopoietin/Tie factors peaked at 96 h, except Ang-2, which gradually increased and peaked at 168 h. CONCLUSIONS: During liver regeneration, hepatocyte proliferation was followed by endothelial cell proliferation. The VEGF family and angiopoietin/Tie system may have distinct roles in angiogenesis, with an enhanced expression of the VEGF family in the early phase of regeneration followed by angiopoietin/Tie expression.

Preventive effect of FK143, a 5alpha-reductase inhibitor, on chemical hepatocarcinogenesis in rats.

The incidence of hepatocellular carcinoma (HCC) is more prevalent in males than in females. 5alpha-Dihydrotestosterone is the most potent form of androgen and is converted from testosterone by 5alpha-reductase. The antitumor effect of a 5alpha-reductase inhibitor (FK143) was evaluated in a rat chemical hepatocarcinogenesis model (Solt-Farber). Male Fischer 344 rats were used in three groups: (a) control group; (b) low-dose FK143 (FKL) group (20 ppm FK143); and (c) high-dose FK143 (FKH) group (200 ppm FK143). The numbers of both glutathione S-transferase placental form-positive foci (P < 0.05) and hyperplastic nodules (HNs; P < 0.01) in the liver were significantly lower in the FKL group than in the control group. The numbers (P < 0.05) and tumor volume (P < 0.01) of HCCs per liver were significantly lower in the FKL group when compared with the control group. All HCCs were well differentiated in the FKL group, whereas 38% and 36% of HCCs were moderate to poorly differentiated in the control group and the FKH group, respectively. The proliferating cell nuclear antigen labeling index:apoptotic index ratios of enzyme-altered foci, HNs, and HCCs were significantly lower in the FKL group than in the control group. Serum 5alpha-dihydrotestosterone was significantly lower in both the FKL and FKH groups. However, a high dose of FK143 (200 ppm) provided no protection against hepatocarcinogenesis, and the level of serum testosterone was elevated in this group when compared with that in the control group. The low dose of FK143 significantly suppressed the formation of enzyme-altered foci, HNs, and HCCs in rat hepatocarcinogenesis. This may indicate that 5alpha-dihydrotestosterone enhances hepatocarcinogenesis. We conclude that an optimal dose of FK143 may have a suppressive effect on hepatocarcinogenesis.

The clinicopathological significance of heparanase and basic fibroblast growth factor expressions in hepatocellular carcinoma.

Heparan sulfate plays an essential role for insolubility of the components of extracellular matrix and represents a storage depot for various growth factors. Therefore, heparanase produced by a given tumor may facilitate tumor invasiveness and angiogenesis through the release of heparan sulfate-bound growth factors. Although the growth factors responsible for angiogenesis in hepatocellular carcinoma (HCC) have recently been investigated, the clinicopathological significance of heparanase in connection with basic fibroblast growth factor (bFGF) expression in HCC has not been evaluated so far. Fifty-five patients who had undergone hepatic resection for HCC without preoperative treatment were included in the present study. Expression of heparanase mRNA was evaluated by reverse transcription-PCR, and bFGF was examined by Western blotting using a monoclonal antibody. Tumor angiogenesis was evaluated by immunostaining with a factor VIII-related monoclonal antibody. Expression of heparanase mRNA was detected in 47% of HCCs and was significantly correlated with larger tumor size (P = 0.01), presence of portal vein invasion (P = 0.01), and higher overall tumor invasiveness (P = 0.02). Moreover, its expression was correlated with tumor microvessel density (MVD; P = 0.02). There was a direct correlation between the levels of bFGF proteins and MVD in HCCs (P = 0.0001), and, furthermore, concomitant expression of bFGF and heparanase was associated with higher tumor MVD as compared with expression of either factor alone (P = 0.01). In conclusion, the expression of heparanase in HCC enhances growth, invasion, and angiogenesis of the tumor, and bFGF seems to be a potent angiogenic factor for HCC.

Prognostic factors and survival after hepatic resection for hepatocellular carcinoma without cirrhosis.

BACKGROUND: Detailed follow-up of patients with chronic hepatitis has resulted in increased diagnosis of hepatocellular carcinoma (HCC) in patients without cirrhosis. Despite numerous studies on hepatic resection, the prognostic factors for intrahepatic recurrence and survival are not well known for patients with HCC without cirrhosis. METHODS: Among 349 patients with HCC treated in the past 13 years, cirrhosis was absent in 126 patients (36 per cent). Curative hepatic resection was carried out in 100 (79 per cent) of these patients. Risk factors for intrahepatic recurrence and prognostic factors for survival were evaluated by univariate and multivariate analyses. RESULTS: Postoperative morbidity and mortality rates were 22 and 3 per cent respectively. The 5- and 10-year disease-free and overall survival rates were 31 and 50 per cent, and 22 and 47 per cent respectively. Blood loss, surgical resection margin, intrahepatic metastasis, portal vein invasion and extent of hepatic resection were independently associated with overall survival. However, the only risk factors for intrahepatic recurrence were portal vein invasion and hepatitis C virus (HCV) infection. The former was related to early recurrence while the latter was related to later recurrence. The 5-year disease-free survival rate was 58 per cent in patients with hepatitis B virus infection while it was 6 per cent in patients with HCV infection (P < 0.001). CONCLUSION: In the treatment of HCC without cirrhosis, major hepatectomy is advocated to prevent early recurrence. Liver transplantation may be required for patients with HCV infection.

The influence of perioperative blood transfusion on intrahepatic recurrence after curative resection of hepatocellular carcinoma.

OBJECTIVE: This study retrospectively evaluated the association between perioperative blood transfusion and intrahepatic recurrence in patients with hepatocellular carcinoma (HCC) who had undergone curative hepatic resections. METHODS: Hepatic resection was performed with curative intent in 195 patients with primary HCC between 1985 and 1996. Patients who had received perioperative blood transfusion (transfused group: n = 117) and those who had no perioperative blood transfusion (nontransfused group: n = 78) were compared in terms of conventional prognostic variables and cancer-free survival by the univariate and multivariate analyses. RESULTS: The 1-, 3-, and 5-yr cancer-free survival rates in the nontransfused and transfused groups were 83.4% and 67.9%, 43.0% and 36.7%, and 23.1% and 24.6%, respectively (p = 0.175). Multivariate analysis of prognostic factors in all patients revealed that vascular invasion, tumor size (> or =5 cm), and Child's class were independent factors for intrahepatic recurrence. Further analyses in various stratified groups showed that perioperative blood transfusion was an independent predictor of prognosis in HCC patients with portal vein invasion (RR: 2.8, p = 0.0038). The 1-, 3-, and 5-yr survival rates in the nontransfused and transfused groups with portal vein invasion were 71.9% and 41.6%, 54.5% and 10.9%, and 26% and 0%, respectively (p = 0.0003). CONCLUSIONS: We conclude that perioperative blood transfusions enhance the risk of intrahepatic recurrence of HCC in patients with portal vein invasion. As well, the more difficult surgery and the increased manipulation of the liver that occur in these cases create a greater possibility of tumor dissemination.

Major compared with limited hepatic resection for hepatocellular carcinoma without underlying cirrhosis: a retrospective analysis.

OBJECTIVE: To find out if patients with hepatocellular carcinoma (HCC) with no underlying cirrhosis benefit from major hepatic resection. DESIGN: Retrospective study. SETTING: University hospital, Japan. PATIENTS: 58 patients without cirrhosis and with HCC 10 cm in diameter or less. INTERVENTIONS: 25 had major and 33 had limited hepatic resections. MAIN OUTCOME MEASURES: Overall and disease-free survival, and prognostic factors verified by univariate and multivariate analyses. RESULTS: 6 patients developed major complications (10%), two of whom died within 60 days of operation. There were no differences in postoperative morbidity and mortality between the two groups. The overall and disease-free survival were similar as was the incidence and pattern of intrahepatic tumour recurrence. Hepatitis B surface (HBs) antigen (positive), tumour size (smaller than 3 cm), and surgical margin (clear) were favourable indicators of disease-free survival on multivariate analysis. CONCLUSIONS: Major hepatic resection should not necessarily be done for HCC without cirrhosis but it is important to take an adequate surgical margin. Overall and disease-free survival are better in patients who are HBs-antigen positive than those who are negative because most of the latter are positive for hepatitis C virus.

Prognostic factors after hepatic resection for hepatocellular carcinoma associated with Child-Turcotte class B and C cirrhosis.

OBJECTIVE: To evaluate prognostic factors after resection of hepatocellular carcinoma (HCC) in patients with Child-Turcotte class B and C cirrhosis. SUMMARY BACKGROUND DATA: Although hepatic resection remains the mainstay in the treatment of HCC and can be performed with low morbidity and mortality rates in patients without cirrhosis, its role is poorly defined for patients with severe cirrhosis. METHODS: From 1986 to 1996, partial hepatectomy was performed for HCC in 63 patients with Child-Turcotte class B (n = 46) and C (n = 17) cirrhosis. There were 46 men and 17 women, with an average age of 61.2 years (range 35 to 79 years). Associated conditions were diabetes mellitus in 45, esophageal varices in 32, severe hypersplenism in 26, cholelithiasis in 13, gastroduodenal ulcer in 6, and hiatal hernia, gastric lymphoma, splenic abscess, and pancreatic cyst each in 1. Concomitant surgical procedures were performed for most of these conditions. RESULTS: Major complications occurred in 17 patients (27%), six (9.5%) of whom died within 1 month after surgery. The overall in-hospital death rate was 14.3%. Liver failure and intraabdominal sepsis were mostly fatal complications. The overall and disease-free survival rates, respectively, were 70.2% and 64.5% at 1 year, 43.5% and 23.8% at 3 years, and 21.4% and 14.9% at 5 years. Multivariate analysis with the Cox regression model revealed that favorable factors for survival were Child class B, no transcatheter arterial embolization before surgery, young age, and low alanine aminotransferase (ALT) level before surgery. CONCLUSIONS: Hepatic resection can provide a favorable result in young patients with HCC complicating Child class B cirrhosis with low hepatitis activity, but transcatheter arterial embolization before surgery should be avoided in such patients.

Suppressed endothelin-1 production by FK506 and cyclosporin A in ischemia/reperfusion of rat small intestine.

BACKGROUND: Endothelin-1 (ET-1), a novel vasoconstrictor, possibly plays a role in the mediation of ischemia/reperfusion (I/R) injury. Tacrolimus (FK506) and cyclosporin A (CsA) were reported to maintain tissue microcirculation of the liver subjected to I/R. This study investigated the effects of these immunosuppressants on intestinal I/R in terms of intestinal tissue microcirculation associated with ET-1. METHODS AND RESULTS: Male S-D rats were pretreated twice with FK506 (0.2 mg/kg), CsA (10 mg/kg) or only saline solution (0.5 mL). The tissue microcirculation in the control was reduced after I/R (29% +/- 10%) accompanied by hypotension, increased tissue ET-1 expression (25.0% +/- 6.4% to 67.9% +/- 5.0% 60 minutes after reperfusion), and increased ET-1 level in the portal blood (3.4 +/- 0.9 to 23.6 +/- 6.1 pg/mL). FK506 suppressed ET-1 expression (27.3% +/- 5.2%, 4.1 +/- 2.2 pg/mL), maintained microcirculation (96% +/- 16%), and blood pressure, reduced histologic damage, and improved survival. CsA had a similar but weaker effect compared with FK506. An additional experiment was performed with BQ485Na (BQ), an ETA receptor antagonist, to evaluate the genuine role of ET-1. BQ showed almost the same effects as FK506. CONCLUSIONS: FK506 and CsA, particularly the former, maintain microcirculation and protect the tissue from I/R injury by suppressing the production and release of ET-1.

Thymidine phosphorylase (platelet-derived endothelial cell growth factor), microvessel density and clinical outcome in hepatocellular carcinoma.

BACKGROUND/AIMS: Angiogenesis plays an important role in tumor growth and metastasis. It is regulated by angiogenic factors. Thymidine phosphorylase (platelet-derived endothelial cell growth factor) is one such factor. Although the significance of platelet-derived endothelial cell growth factor has been studied for several types of tumor, the expression of platelet-derived endothelial cell growth factor and its correlation with microvessel density or clinicopathological factors in hepatocellular carcinoma are unknown. We evaluated microvessel density and platelet-derived endothelial cell growth factor expression in hepatocellular carcinoma to determine whether microvessel density and platelet-derived endothelial cell growth factor expression are correlated with the clinicopathological factors of hepatocellular carcinoma. METHODS: Using immunohistochemical staining with anti-platelet-derived endothelial cell growth factor antibody and the ELISA method, we evaluated the correlation among platelet-derived endothelial cell growth factor expression, microvessel density and clinicopathological factors in 84 hepatocellular carcinoma patients. Microvessels were stained with anti-human von Willebrand factor (anti-Factor VIII) and anti-CD34. RESULTS: In the surrounding liver, there was a significant correlation between microvessel density and platelet-derived endothelial cell growth factor expression (p=0.002), and hepatitis C virus-positive livers had higher microvessel densities than otherwise (p=0.003). However, this correlation was not found for hepatocellular carcinoma, but hepatitis C virus-positive tumors had higher expression of platelet-derived endothelial cell growth factor (p=0.018). Microvessel density in hepatocellular carcinoma obtained by Factor VIII staining inversely affected the recurrence-free survival rate (p=0.0416), but the microvessel density by CD34 staining was not a significant predictor. CONCLUSIONS: This study indicates that platelet-derived endothelial cell growth factor may not be a major regulator of angiogenesis of hepatocellular carcinoma, but this enzyme may play an important role in hepatocarcinogenesis cooperating with hepatitis C virus. Also, the density, not of sinusoid-like vessels, but of larger vessels in hepatocellular carcinoma could be a prognostic factor for hepatocellular carcinoma.

Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver.

As in other tumors, the assessment of microvessel density (MVD) in hepatocellular carcinoma (HCC) may be essential to perform an effective anti-angiogenic therapy for this tumor. The relationship between vascular endothelial growth factor (VEGF) and MVD of HCC as well as the surrounding liver remains to be elucidated. In 71 patients who had undergone curative hepatic resection for HCC, MVD and VEGF expressions were evaluated for HCC and the liver by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and/or immunostaining. The intensity and extent of VEGF immunoreactivity were evaluated using a computer image analyzer-cell analysis system (CAS). Angiographic data were re-evaluated and compared with MVD in 50 tumors. Tumoral MVD was significantly correlated with tumor capsule formation (t test, P = .0016). Small HCCs (< or = 2 cm) had a significantly lower MVD compared with moderate-sized HCCs (2-5 cm) (t test, P = .016), and the MVD of large HCCs was relatively lower than that of moderate tumors. Tumor vascularity on angiography was not correlated with the MVD. Neither VEGF mRNA levels nor protein expression in HCC were correlated with the tumoral MVD or any histopathological features of the tumor. However, cirrhotic livers had significantly higher MVD and VEGF expressions compared with noncirrhotic livers (t test, P = .0015 and P = .047, respectively). Only the MVD of tumor was significantly correlated with intrahepatic recurrence (t test, P = .0048) and disease-free survival (DFS) rates (log rank test, P = .0035). Moreover, the MVD was an independent predictor for DFS by multivariate analysis (chi2 test, P = .03). In conclusion, the MVD in HCC may be involved in the dismal prognosis of this tumor, and VEGF may be associated with the angiogenic process of the cirrhotic liver, but not with the angiogenesis of HCC.

Proposal of invasiveness score to predict recurrence and survival after curative hepatic resection for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most malignant human tumors and is associated with a high incidence of postoperative recurrence. There is no generally accepted definition for HCC invasiveness. Moreover, the predictive value of the pathologic factors that reflect HCC invasiveness was previously studied as separate events, with much controversy among different study groups. In this study, we proposed an invasiveness scoring system based on the relative importance of six criteria for HCC invasiveness: portal vein invasion, intrahepatic metastasis, hepatic vein invasion, serosal invasion, absence of tumor capsule, or presence of capsular invasion. METHODS: A total of 137 patients (111 male and 26 female) who underwent curative hepatectomy for HCC were included. Scoring of the six pathologic parameters was based on the clinical significance of each parameter as a single predictor for recurrence after curative resection. According to our scoring system, the patients were divided into three groups: low invasive HCC group A with a total invasiveness score 0 to 1, moderately invasive group B with a score of 2 to 4, and highly invasive group C with a total score of 5 or greater (5 to 11 points). RESULTS: Evaluation of the current scoring system showed a significant stepwise increase in the incidence of recurrence as the invasiveness score increased. Moreover, disease-free survival was significantly different among the three groups (log rank p < 0.0001). The 1-, 3-, 5-, and 8-year disease-free survival rates were 89%, 59%, 54%, and 54% in group A; 72%, 32%, 12%, and 10% in group B; and 54%, 19%, 7%, and 0 in group C, respectively. Multivariate analysis showed that the patients of groups B and C had a significantly worse prognosis compared with those of group A (p < 0.0001). CONCLUSIONS: The current scoring system can classify HCCs into three invasive categories and predict more accurately recurrence and disease-free survival after curative hepatectomy compared with any single invasive parameter previously proposed. Moreover, this system can be used as a therapeutic guide during and after the surgical decision making.