Increased Migration And Invasion Capabilities Of Endothelial Cells Derived From Juvenile Nasopharyngeal Angiofibroma Compared With Normal Endothelial Cells

Juvenile nasopharyngeal angiofibroma (JNA) which constitutes～0.5% of all head and neck tumors is described as an uncommon vascular tumor that is diagnosed typically in the adolescent male patient as a nonencapsulated, submucosal spreading tumor.Although histologically benign in appearance, JNAs are locally aggressive and destructive, spreading from the nasal cavity to the nasopharynx, paranasal sinuses, and orbit skull base with intracranial extension. Without established pharmacological treatments or risk-reduced radiological therapies, surgery is unavoidable for patients with advanced JNA. Although recent medical technologies have provided new options and treatment courses for the ENT physician, especially the use of embolization and transnasal endoscopy, the recurrence rate following surgical treatment of JNA is still up to 50%, which is the major obstacle to further improve the prognosis of JNA patients. Therefore it is urgently needed to investigate the factors and its molecular basis for recurrence. Only in this way, better prognosis might be further achieved.Though the etiology and pathogenesis of JNA remain unknown, vascular origin is one of the main theories of its origin. Angiogenesis is an essential process required for growth and metastasis in solid tumors, which is similar in JNA as a highly vascularized angiofibroma. However there is little research focused on vascular endothelial function of JNA, especially the endothelial cells (ECs) themselves.In the present study, we analyzed the expression of CD 105 in microvessel endothelial cells using tissue microarray based immunohistochemistry and its prognostic significance in JNA. Meanwhile we isolated, cultured, passaged, and characterized the phenotypic and functional properties of JNA-derived endothelial cells (JECs), then we compared CD105+ JECs with human umbilical vein endothelial cells (HUVECs) by examining their abilities of proliferation, motility, invasion, in order to elucidate the reasons and molecular basis for high recurrence of JNA and its value as a therapeutic target, which are the aims of this study. PART ONECD105 expression of microvessel endothelial cells in JNA and its prognostic significance.Purpose:To investigate CD105 expression of microvessel endothelial cells in JNA and its relationship with the recurrence of JNA.Methods:We applied the method of immunohistochemistry to detect the expression of CD 105 in an independent tissue microarray (70 patients diagnosed with JNA), and analyze the correlation between CD105 expression of microvessel endothelial cells and clinicopathological features, time to recurrence (TTR)Results:Immunohistochemical results showed that the expression of CD 105 was in endothelial cell but not in stroma of JNA. Chi-Square test suggested microvessel density (MVD) had correlation with the recurrence of JNA (P=0.013). TTR of patients with low MVD was significantly higher than patients with high MVD (P =0.009). Multivariate analysis indicated that MVD was a decisive independent prognostic factors of JNA (P=0.01).Conclusions:MVD does relate to the prognosis of JNA, which suggests that angiogenesis may play an important role in the occurrence and development of JNA. Meanwhile, it can be expected to become a new therapeutic targets and prognostic predictor.PART TWOIsolation, characterization and culture of endothelial cells derived from human juvenile nasopharyngeal angiofibromaPurpose:Isolation, characterization and culture of endothelial cells derived from human juvenile nasopharyngeal angiofibroma.Methods:A modified immunomagnetic method using magnetic beads conjugated with anti-CD 105 antibody was used to isolate vascular endothelial cells from human JNA. To identify the purity of sorting cells, Flow cytometry was used. By immunofluorescence staining of von Willebrand factor (vWF), internalization of acetylated low-density lipoprotein (Ac-LDL) and tube formation test, characterization of the isolated cells were distinguished.Results:The isolated JEC didn’t present typical "cobblestone" appearance. The expression of CD105 and von Willebrand factor (vWF) was positive in more than 99% of isolated cells. Internalization of acetylated low-density lipoprotein was positive in more than 95% of isolated cells. The isolated cells could form capillary-like tubes on BD Matrigel Matrix.Conclusions:JECs can be isolated from JNA using modified immunomagnetic method, and these cells can be serially subcultured.PART THREEFunctional characteristics of endothelial cells derived from juvenile nasopharyngeal angiofibromaPurpose:To compare the function distinction of CD105+ JECs with HUVECs and to investigate the possible molecular basis.Methods:Wound healing assay, transwell assay, immunofluorescence staining of F-actin and western blot had been done to compare the distinction between CD105+ JECs and HUVECs.Results:Wound healing assay revealed increased migration ability of CD105+ JECs and transwell assay revealed increased invasion ability of CD105+ JECs, with respect to HUVECs. Western blot analysis revealed increased expression of VEGFR1, p-MAPK, MMP-2 in CD105+ JECs than HUVEC, but decreased expression of VEGFR2, p-ERK.Conclusions:CD105+ JECs display increased migration and invasion activity, which may attribute to the VEGFR pathway.