Expression Of Endostatin, VEGF And Their Relationship To Angiogenesis In Oral Leukoplakia And Oral Squamous Cell Carcinoma

Oral leukoplakia (OLK) assessed histopathologically as epithelial unusual hyperplasias is the most common premalignant lesion in oral cavity. A total of 3%~5% patients with OLK will develop into oral squamous cell carcinoma (OSCC). OSCC is the leading malignant tumor in oral cavity and accounts for more than 80% of the oral malignant tumor. It is generally accepted the concept that the process of OLK to OSCC is a multistage and multi-step which arouses the attentions of academia.In recent years, studies suggested that the induction of angiogenesis is an earlier event of carcinogenesis and an important step in this process which regulated by proangiogenic and antiangiogenic factors. Angiogenesis plays an important role in carcinogenesis of premalignant lesion and progression of tumor. The study on the proangiogenic and antiangiogenic factors of tumor angiogenesis is becoming another hotspot. Endostatin is an endogenous angiogenesis inhibitor that was first isolated from the supernatant of an in vitro culture of EOMA cells, a murine hemangioendothelioma cell line. Further characterization revealed that Endostatin is a 20-KD C- terminal fragment of collagen ⅩⅧ . In vitro culture, human and murine Endostatin specifically inhibit the proliferation and migration of capillary endothelial cells and can induce apoptosis of proliferating endothelial cells. Vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF) has been popularly accepted as the most potent angiogenic factor in tumor angiogenesis. It is a glycoprotein ofabout 45-KD, which can promote the proliferation of endothelial cells and new blood vessel forming, increase vessel permeability, and play a key role in the maintenance of normal situation and the occurrence and development of tumor. Microvascular density (MVD), a measure of tumor angiogenesis was identified by immunostaining of endothelial cells using anti-CD34 antibody. At present, less is known about study on the expression of Endostatin, VEGF as well as their relationship with angiogenesis in OLK and OSCC.Objective The purpose of this study aimed at investigating the expression ofEndostatin, VEGF and CD34 as well as their relationship with angiogenesis in OLK and OSCC in order to evaluate their roles in carcinogenesis and progression of OSCC, and to provide a potential new way for early diagnosis, prognosis assessment and indicating theraputics of OSCC.Materials and methods1. A total of 42 OLK specimens, 36 OSCC specimens and 10 normal mucosa (NM) tissue specimens were collected. The tissues were fixed in formalin and embedded in paraffin. The OLK specimens were assessed histopathologically as simple hyperplasia (13cases), mild dysplasia (1 leases), moderate dysplasia (lOcases) and severe dysplasia (8cases), respectively. The OSCC specimens were pathologically classified as 14 of grade I, 12 of grade II, and 10 of grade III. The NM tissue specimens were treated as control.2. Immunohistochemical staining was employed to detect the expression of Endostatin, VEGF and CD34 in all specimens. The expression of positive cells was described as grey value using the analysis of immunohistochemical semi-quantitate performed with a computerized microscopic image processor.3. The results were analyzed using Chi-square, Fisher's exact test, analysis of variance (ANOVA) and Mest by SPSS 10.0 software. Statistically significant level was considered as "alpha equals 0.05".Results1. The positive staining for Endostatin protein was mainly observed in the cytoplasm of cancer cell and endothelium cells of blood vessel. Its positive rate in NM, simple hyperplasia OLK, dysplasia OLK and OSCC were 20.00%, 38.46%, 55.17% and 77.78%, respectively.Significant differences were found between OSCC and other groups (PO.05). The average grey value which indicated positive staining of Endostatin were significantly higher among the groups except inNM and simple hyperplasia OLK (PO.05).2. The positive staining for VEGF protein was mainly observed in the cytoplasm of cancer cell. Its positive rate in NM, simple hyperplasia OLK, dysplasia OLK and OSCC were 20.00%, 53.85%, 68.97% and 83.33%, respectively. The result showed a significant difference existed among the four groups (P<0.05). The average grey value of VEGF were significantly higher among the groups (PO.05). The positive rates and grey value of VEGF in grade I, II, III of OSCC showed no significant differences (P>0.05).3. The MVD was immunohistochemically stained with anti-CD34 antibody. The positive staining for CD34 was mainly localized in the cytoplasm of vascular endothelial cell. The MVD described as mean ±standard deviation in NM, simple hyperplasia OLK, dysplasia OLK and OSCC were 9.00±5.72> 13.92±7.18> 20.41±9.25 and 29.56±10.74, respectively. The data showed a significant difference among the groups except NM and simple hyperplasia 0LK(PO.05).In grade I, II, III of OSCC groups, the mean MVD showed the statistical significances between grade I and III, grade II and III of OSCC groups (PO.05) .4. In all specimens, there was a significant correlation between expression of Endostatin and VEGF protein (PO.05) .5. In OSCC, the MVD in Endostatin positive group(27.50± 10.35) was lower than in Endostatin negative group( 36.75±9.38), and there was a significant difference between them(PO.05) .6. In all specimens, the MVD in VEGF positive group and VEGF negative group were 25.90±10.79 and 13.76±9.25, respectively, and there was a significant difference between them (PO.05) .Conclusion1. The increased expression of Endostatin and VEGF protein according to the process of NM to OLK and OSCC, suggesting that Endostatin and VEGF may take part in the carcinogenesis of OLK and play an important role in the progression of OSCC. Theincreased expression of Endostatin may be considered as a state of protective reaction against blood vessel forming of tumor.2. The increasing of MVD with carcinogenesis of OLK and the degree of differentiation in OSCC identified that the induction of angiogenesis is an earlier event of carcinogenesis. MVD may be an important marker in evaluating angiogenesis and the biological character of OSCC.3. There is a significant correlation between expression of Endostatin and VEGF protein in the carcinogenesis of OLK, suggesting that they played a cooperative role in angiogenesis and participated in the carcinogenesis and progression of OSCC.4. In OSCC, the result that MVD in Endostatin positive group is lower than in Endostatin negative group consistent with the hypothesis that Endostatin as an endogenous angiogenesis inhibitor regulates the angiogenesis of tumor.5. The combined detecting of the expression of VEGF, Endostatin and MVD by imrnunohistochemistry staining contributes to the early diagnosis, therapeutics and prognostic assessment of OLK.