Hepatocellular carcinoma (HCC) is a hypervascular tumor whose growth and metastasis are angiogenesis dependent. Angiogenesis, the formation of new blood vessels, is essential for tumor growth by providing nutrients and eliminating metabolic waste products.1,2 Tumor angiogenesis and its clinical significance have been studied in a great variety of neoplasms. The factors that regulate tumor angiogenesis include promoters and inhibitors. The growth and metastasis of tumors can be stimulated when promoters are strong or inhibitors are weak. Otherwise, tumors' growth and metastasis would be inhibited.3
Endothelial cells are the most important part of tumor angiogenesis. Tumor blood vessels in HCC are largely derived from the sinusoidal endothelial cells (SECs). SE-1 antibody has been established as an antibody that reacts specifically with SECs of the adult rat liver but not with any other type of vascular endothelial cells.4 Therefore, SE-1 antigen may be a useful marker to study the role of hepatic SECs or endothelium in various pathophysiological conditions of the liver.5 The relationship between SE-1 antigen and HCC and angiogenesis has not been reported, neither has the SE-1 antigen expression in serum been reported.
CD31 and CD105 are also endothelial markers6 for HCC. The purpose of this study was to investigate the relationship between SE-1 and HCC, using CD31 and CD105 as controls. Immunohistochemistry (IHC) was used to study SE-1, CD31 and CD105 expressions in liver SECs and in tumor endothelial cells (TECs) of HCC. In addition, the study used ELISA to compare the titers in serum in rats bearing HCC with normal rats to build the basis of the study of HCC in molecular biology.
Rats were maintained in a temperature-controlled room under 12 hours day-night rhythm with free access to water and a standard rat diet. The McA-RH 7777 (ATCC, USA) cell line was used to induce the liver tumor. One million McA-RH 7777 cells were inoculated into the liver lobe7 of 8-week old male Buffalo rats and tumors were harvested after 28 days. For the immunohistochemistry study, tumors of 2.5 cm-3.0 cm in diameter were harvested. Venous blood was collected from 20 Buffalo rats with HCC and 18 normal Buffalo rats. Serum was collected after centrifugation and was stored at -80°C until use.
The Table details the characteristics, pretreatments, localizations, and sources of the three antibodies used in the study.
The tissues were fixed in 10% neutral buffered formalin for 24 hours-48 hours, then dehydrated in graded alcohol and histoclear followed by paraffin embedding. Paraffin blocks were sectioned at 4 μm. Sections were de-waxed in histoclear, and rehydrated in graded alcohol. For antigen retrieval, sections intended to be stained by anti-CD31 antibody were heated at 98°C for 20 minutes in 10 mmol/L citrate buffer, pH 6.0. The sections for the rest of antibodies were incubated with 0.1% pronase E at room temperature for 10 minutes. All sections were soaked in 3% hydrogen peroxide solution for 25 minutes to counteract endogenous peroxidase activity. After blocking with 5% BSA in PBST at room temperature for 30 minutes, the rabbit polyclonal antibodies against CD31, the rat polyclonal antibodies against CD105 and the mouse monoclonal SE-1 antibody were applied to the slides at dilutions of 1:500, 1:200 and 1:20, respectively. After incubation at 4°C overnight, secondary antibody was applied for one hour at room temperature. The color was developed by reacting with DAB substrate (DAKO, Germany). Slides were then counterstained with hematoxylin, dehydrated, cleared, and mounted.
The differences between CD31, CD105 and SE-1 titers in HCC rat sera and normal rat sera were tested by ELISA. Ninety-six-well microtiter plates were coated with serum in PBS at 4°C overnight and were blocked with 3% BSA in PBST for 1 hour at 37°C. The plates were incubated with anti-CD31 antibody, anti-CD105 antibody and the SE-1 antibody followed by IgG-HRP reagent for 1 hour. A color reaction was induced by the addition of premixed TMB substrate solution (DAKO) and was stopped 30 minutes later by the addition of 1 mol/L H2SO4. The absorbance was measured at OD450 nm using a Spectra Max 100 ELISA reader (Molecular Devices, USA). Analysis used the method of Tian et al.8
Statistical analysis was carried out using SPSS 13.0 software. Values were expressed as mean ± standard deviation (SD). Student's t test was performed to analyze the difference between the groups. The heterogeneity of variance was evaluated using Satterthwaite's test. A P value >0.01 was statistically significant in comparison to the control group.
Differential expressions of SE-1, CD31 and CD105 in tumor endothelial cells and sinusoidal endothelial cells. All three antibodies stained endothelial cells specifically but with different patterns (Figure 1). Expression of SE-1 antigen in HCC was different from the expression of CD31 and CD105. As shown in Figure 1, in the tumor boundary SE-1 stained the endothelial cells of blood vessels both in tissue adjacent to the tumor and in the liver tumor. A difference was that SE-1 exhibited stronger staining in tissue adjacent to the tumor than in the tumor tissue. In the tumor center, less SE-1 was seen in the blood vessels compared with SE-1 found in the boundary of the tumor.
Compared with normal liver tissue tumors having few expression of SE-1, expressions of CD31 and CD105 in normal liver tissue were less than those in liver tumors (Figure 1). CD31 showed only focal reactivity in a few sinusoids in normal liver but showed strong staining in tumor endothelial cells. In the tumor center there was a moderate increase in the number of CD105 positively stained blood vessels compared with the tumor boundary (Figure 1).
CD105 stained microvessels in and around the tumor. Both tumor endothelial cells and normal liver endothelial cells showed staining of CD105, but in the tumor the staining was stronger. In normal liver tissue, SE-1 had higher expression than in tumor tissue, while expressions of CD31 and CD105 in normal liver tissue were less than those in tumor tissue (Figure 1).
Serum SE-1, CD31 and CD105 antigen in rats with HCC and normal rats
The mean optical density of the three holes was determined (D450 nm). Standard deviation of the hole centre distance was lower than 0.024 and coefficient of variation was less than 9.8%, which indicated that both the measurement system and the operation were reliable.
As shown in Figure 2, the levels of serum SE-1 antigen in rats with HCC were significantly lower than those in healthy subjects (D450 nm: 0.908±0.191 vs. 1.270±0.403, t=3.4983, P=0.0011). Serum levels of CD31 and CD105 in rats with HCC were significantly elevated, compared with the controls (D450 nm: 0.165±0.058 vs. 0.122±0.033, t=2.8628, P <0.0086, for CD31; 2.349±0.411 vs. 1.729±0.449, t=4.4922, P>0.0001, for CD105) (Figure 2).
The process of tumor growth and metastasis is dependent on angiogenesis as had been studied intensively by Folkman from the 1970's. After this, a large number of substances which will interfere with the process of angiogenesis have been identified.9-11When the balance between promoters and inhibitors is broken, the switch to angiogenesis is promoted12-14 and new blood vessels are formed for tumor growth and metastasis. A few angiogenic factors have been reported to promote angiogenesis in HCC, but the detailed mechanisms of HCC angiogenesis are still largely unknown.
We investigated whether SE-1, that specifically recognizes a 45-KD antigen expressed only in rat liver SEC but not any other type of endothelial cell, is related to HCC. Our results showed that all three tested markers, SE-1, CD31 and CD105, were expressed by endothelial cells in tumor-associated vessels of hepatocellular carcinoma. SE-1 binds an antigen, the nature and function of which is yet to be known. The antigen is thought to be related to the specific function of SEC in adult rat livers.
Judging from its cellular localization at the surface of the plasma membrane and inner surface of pinocytotic vesicles, it is postulated to be a receptor molecule which is specifically expressed in SEC.5 Sandra et al15 identified the antigen recognized by SE-1 as CD32b. They considered that SE-1 antibody did not cross-react with mouse and human species. They found that CD32b was a low-affinity Fcγ receptor constitutively expressed by human adult liver sinusoidal endothelial cells that had the same expression pattern as that described for SE-1 in rats. In our study, SE-1 stained the endothelial cells of blood vessels both in non-neoplastic tissue and in the periphery of liver tumors. We observed that more SE-1 antigen was expressed in normal liver than in tumor tissue. In the tumor center there were fewer blood vessels stained by SE-1 antibody compared with the boundary of the tumor. In addition, the levels of serum SE-1 antigen in rats with HCC were significantly decreased compared with healthy rats. This is a relatively early report of the expression of SE-1 and liver cancer. Down regulation of SE-1 in HCC means that SE-1 is closely associated with HCC angiogenesis.
CD31 is a cell-cell adhesion molecule of the immunoglobulin superfamily expressed by most endothelial cells.16 Previous work has shown that CD31 is not detected or only barely detectable on normal SECs. But it is overexpressed along sinusoids in various pathological situations, including tumor, fibrosis and inflammation.17-19 Related studies have shown that the expression of CD105 was up-regulated in many tumors with a poor prognosis, such as endometrial carcinoma, cervical cancer, colorectal cancer and breast carcinoma.20-24 In our study, CD31 was barely detectable on normal SECs but stained strongly in tumor endothelial cells. CD105 stained microvessels in and around the tumor. CD105 stained SECs as well. The levels of serum CD105 in rats with HCC were significantly increased compared with tumor free rats, consistent with Eray Yagmur's work.25 A previous study showed that when the expression of CD105 was inhibited in human umbilical vein endothelial cells, angiogenesis was significantly inhibited. This means that CD105 could participate in angiogenesis and development. Expression of CD31 in serum associated with HCC has not been reported. Our results show that serum CD31 levels in rats with HCC were significantly elevated, compared with those of the controls, which is consistent with the result of IHC. Those results show that there is a close relationship between HCC angiogenesis and CD31.
Based on the results obtained in the experiment, it may safely be concluded that SE-1 is closely related with liver tumor angiogenesis as are CD31 and CD105 arising from their similar expressions in serum. This information may be of great help building the molecular biology basis of HCC's development and contribute to finding a new therapy for liver cancers.
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