| Malignant glioma is the most common and aggressive primary brain tumor. Even after surgical excision, radio and chemotherapy, it shows a high percentage of recurrence and short survival time. Malignant glioma is highly vascularized tumors of human. Chemotherapy is often unsatisfactory because of the presence of blood-brain barrier. It is well known that angiogenesis is a key factor in the progression of malignant tumors. Many efforts have focused on new therapy approaches based on the inhibition of angiogenesis.Anti-angiogenic therapy is efficient and specific, which avoids the blood-brain barrier and the side-effects of traditional therapy. Thus, it is expected to provide a new and more effective way for treatment of brain tumors. Anti-angiogenesis to treat cancer during the past 40 years has been made great progress. Several anti-angiogeneic drugs have been used for clinical trials, however, they showed limited improvement in overall survival.Targeting tumor endothelial cells is now the main strategy for inhibition of angiogenesis. Therefore, the search for specific targets of tumor angiogenesis has been very important. Accumulating evidence has shown that CD105 (endoglin) specifically expressing on vascular endothelial cells participates in tumor angiogenesis. Thus, CD105 might be emerging as a promising target of anti-angiogenetic therapy of cancer. CD105 gene is located on human chromosome 9q34, encoding a dimer of the same type of membrane-bound glycoprotein, and CD105 has molecular weight of 180kD and locates on cytoplasm membrane. CD105 is a component of TGF- receptor complex involved in cardiovascular development, body homeostasis and other physiological processes. CD105 has been identified as one of endothelial markers, and is correlated with tumor grade, stage, metastasis and prognosis. Currently, CD105 in glioma angiogenesis and its mechanism has not been fully elucidated. Cancer stem cells (CSCs), or tumor initiating cells, account for a very small cell population in a tumor and have self-renewal and differentiation capacities. Emerging evidence shows that CSCs may be directly or indirectly involved in the process of tumor angiogenesis. However, the possible interaction between GSCs and endothelia cells (ECs), and transdifferentiated EC phenotype of GSCs have not been clarified.In this study, human glioma specimens were used to study the CD105 protein expression and its relationship with tumor grading and patient survival. Furthermore, fresh glioma specimens were used for endothelial cells and culture of GSCs. In addition, human ECs (HUVE-12 cells) were used for the study of CD105 function. The main results and conclusions are as follows:1. CD105 protein was found expressed in glioma tissues. Microvessel density (CD105- MVD) was statistically different between WHO grades of gliomas, i.e., the higher the grades, the higher the CD105-MVD. More importantly, high CD105-MVD was correlated with poor prognosis of patients. High and low CD105-MVD values in the patients with survival rate were significantly correlated (Log Rank test: 24.27; P <0.001). Thus, CD105 can be a marker for classification of glioma grade to predict prognosis of patients.2. Glioma derived microvascular endothelial cells were isolated from primary human glioma, and were strongly positive for CD105, CD31, CD34, F -R, but without expression of GFAP and -SMA, the marker for the smooth muscle and pericytes. Culture in Matrigel showed spontaneous tubular formation of endothelial cells.3. Tumorspheres were obtained from primary glioma cells, suggesting their features of cancer glioma stem cells (GSCs). Tumorspheres were found to grow into ECs during co-culture and to transdifferentiate into EC phenotype in the presence of endothelial medium.4. Silencing CD105 gene expression showed inhibition of angiogenesis in vitro. By flow cytometry, real-time quantitative PCR and western blot evaluation of lentiviral interference effects. Virus-positive cell rate was 94% measured by flow cytometry. Real-time PCR and western blot showed that CD105mRNA and protein levels in the CD105-silenced cells - shCD105 were significantly lower than shCon and Mock group. AS compared with Mock, the proliferation of shCD105 cells in the presence of VEGF was decreased. Transwell experiment showed significantly reduction in the number of migrating cells (P <0.01), and scratch test revealed inhibited migration (P <0.01). Tubular formation in Matrigel was also significantly reduced (P <0.01). Real-time PCR showed that CD105 gene silencing could reduce the expression of VEGFR2 mRNA and MMP2 mRNA.In summary, our results suggest that CD105 expression with high CD105-MVD values is predictive for poor prognosis of glioma patients, and CD105 protein is a promoting regulator for glioma angiogenesis. Moreover, we found that glioma stem cells could interact with endothelia cells and present EC phenotype to participate in the angiogenesis of glioma.
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