| Backgrounds: Gastric carcinoma is one of the most common malignancies in the world, especially in Eastern Asia including China, South Korea, and Japan. Because of the high detection rate of early cancer and wider implementation of radical surgery, the overall survival in patients with gastric carcinoma has improved. However, the treatment outcome of this common malignancy is still not satisfactory and various chemotherapeutic attempts in an adjuvant setting have failed to improve the survival rate in gastric cancer. Recently, angiogenesis has been related to hematogenous recurrence and poor prognosis in gastric cancer. Angiogenesis, the process leading to the formation of new blood vessels, plays a central role in the survival of cancer cells, in local tumor growth, and in the development of distant metastasis. Massive formation of blood vessels at the tumor site increases the opportunity for tumor cells to enter the circulation. Therefore, microvessel density (MVD) is considered to influence tumor metastasis and consequently prognosis in various human cancers. Physiologically, the body controls angiogenesis through a series of "on" and "off" regulatory switches: the main "on" switches are known as angiogenesis growth factors (cytokines); the main "off" switches are known as endogenous angiogenesis inhibitors. When angiogenic growth factors are produced in excess of angiogenesis inhibitors, the balance is tipped in favor of blood vessel growth. When inhibitors are present in excess of stimulators, angiogenesis is stopped. The normal, healthy body maintains a perfect balance of angiogenesis modulators. The formation of tumor microvessels is stimulated by angiogenic factors such as vascular endothelial growth factor (VEGF), a 45kDa glycoprotein that is mitogenic for endothelial cells. VEGF binds to specific receptors on endothelial cells, where it induces endothelial proliferation and capillary tube formation, enhancing tumor neovascularization or angiogenesis. Endostatin, a 20-kDa, C-terminal fragment of collagen X VIII, is an endogenous specific inhibitor of endothelial cell proliferation and angiogenesis initially isolated from a murine hemangioendothelioma cell line. Endostatin has subsequently been shown to affect a number of other endothelial cell functions implicated in angiogenesis including migration, survival, proteinase activity and vessel stabilization.Objective: To investigate the expression of VEGF, endostatin and MVD in gastric carcinoma and its relationship with clinicopathological features of human gastric carcinoma.Methods: Serum VEGF and endostatin levels were measured by enzyme-linked immunoassay in 60 patients with gastric carcinoma before and after surgical resection (two weeks later) and in control groups including 30 patients with chronic gastritis and 30 healthy persons. Specimens including paired gastric cancer tissues and normal gastric mucosa (at least 5cm far away from the margin of cancer) obtained from 36 patients undergoing surgical resection for gastric cancer were used in the following study. RT-PCR was employed to analysis the gene expression of VEGF and endostatin, immunohistochernistry and Western blotting were used to analysis the protein expression of VEGF and endostatin in these specimens. Tumor MVD was also determined immunohistochemically with anti-CD34 antibody and estimated by image analysis in these surgically resected tissues. SPSS 13.0 software application was used for all analysis and statistical significance was defined as P values less than 0.05.Results:Part 1:1. The preoperative serum levels of VEGF in patients with gastric carcinoma (394.81pg/ml±159.49pg/ml) were significantly higher than that in patients with chronic gastritis (125.90pg/ml±18.09pg/ml) and healthy persons (117.54 pg/ml±25.27 pg/ml)(P<0.01).2. The preoperative serum levels of endostatin in patients with gastric carcinoma (56.05ng/ml±13.86ng/ml) were significantly higher than that in patients with chronic gastritis (34.01ng/ml±4.39ng/ml) and healthy persons (32.82ng/ml±6.71 ng/ml) (P< 0.01).3. There is a significant positive correlation between preoperative serum VEGF and endostatin levels (r = 0.583, P < 0.01).4. Serum VEGF and endostatin levels before operation were closely related to grade of cell differentiation, depth of invasion, regional lymph-node metastasis, distant metastasis and P-TNM stage (P < 0.05), but their relations to sex were not significant (P > 0.05).5. The postoperative serum levels of VEGF (203.28pg/ml±97.31pg/ml) were decreased significantly than that of preoperation (P < 0.01), while postoperative serum endostatin levels (65.96ng/ml±14.33ng/ml) were increased significantly than that of preoperation (P < 0.01).Part 2:1. The mean expression of VEGF mRNA in gastric cancer tissues was significantly higher than that in normal gastric mucosa [(0.7790±0.1871) vs (0.3436±0.1581), P < 0.01], and the mean expression of endostatin mRNA in gastric cancer tissues was also significantly higher than that in normal gastric mucosa [(0.6735±0.1603)vs(0.5424±0.1625),P<0.01].2. Increased abundance of VEGF and Endostatin mRNA correlated significantly with depth of invasion, regional lymph-node metastasis, distant metastasis and P-TNM stage (P < 0.05), but their relations to the grade of cell differentiation and sex were not significant (P > 0.05).3. There is a significantly positive correlation between abundant VEGF mRNA and endostatin mRNA in gastric cancer tissues (r = 0.613, P < 0.01).4. By immunohistochemistry, the positive rate of expression of VEGF protein in gastric cancer tissues was significantly higher than that in normal gastric mucosa (80.56% vs 22.22%, x~2= 24.519, P < 0.01), and the positive rate of expression of endostatin protein in gastric cancer tissues was significantly higher than that in normal gastric mucosa (61.11% vs 25.00%, x~2 = 9.574, P < 0.01), too.5. By immunohistochemistry, VEGF and endostatin protein expressions were closely related to depth of invasion, regional lymph-node metastasis and clinicopathological stage (P < 0.05), and endostatin but not VEGF was associated with distant metastasis. Neither VEGF nor endostatin correlated with sex and grade of cell differentiation (P > 0.05).6. By immunohistochemistry, the MVD in gastric cancer tissues was significantly greater than that in normal gastric mucosa [(43.67±8.59) vs (12.14±6.08), P < 0.01]. MVD correlated significantly with depth of invasion, regional lymph-node metastasis, distant metastasis and P-TNM stage (P < 0.05), but not with sex and grade of cell differentiation (P>0.05).7. In Western blotting, the expression of VEGF protein in gastric cancer tissues was significantly abundant than that in normal gastric mucosa [(0.6065±0.1805) vs (0.3380±0.1527), P < 0.01], and the expression of endostatin protein in gastric cancer tissues was also significantly abundant than that in normal gastric mucosa [(0.6000±0.1524) vs (0.3481±0.1480), P < 0.01].8. In Western blotting, there is a significantly positive correlation between abundant VEGF protein and endostatin protein in gastric cancer (r = 0.581, P < 0.01).9. In Western blotting, VEGF and endostatin protein expressions were closely related to depth of invasion, regional lymph-node metastasis, distant metastasis and P-TNM stage (P < 0.05), but not to sex and grade of cell differentiation (P > 0.05).10. In gastric cancer, there is a significantly positive correlation between MVD and VEGF (including VEGF mRNA and protein), but the corrlelation between MVD and endostatin (including endostatin mRNA and protein) was not significant.Conclusions:1. There is excessive expression of VEGF and endostatin in gastric cancer tissues, leading to high serum VEGF and endostatin levels.2. Homo-expression of VEGF and endostatin closely correlated with depth of invasion, regional lymph-node metastasis, distant metastasis and P-TNM stage and may be used to evaluate the biological behavior and predict invasion and metastasis of gastric carcinoma.3. MVD is significantly higher in gastric cancer tissues than in normal gastric mucosa and closely correlated with depth of invasion, regional lymph-node metastasis, distant metastasis and P-TNM stage. MVD is one of the important predictors of the biological behavior in gastric carcinoma.4. There is a significantly positive correlation between abundant VEGF and endostatin in gastric cancer. Both VEGF and endostatin play an important role in the neoangiogenesis of human gastric cancer.5. VEGF and endostatin may be important targets for antiangiogenesis therapy in patients with gastric carcinoma.
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