טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentFink-Barkai Dorit
SubjectThe Role of NrCAM in Endothelial Cells Biology and
Angiogenesis
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Gera Neufeld
Full Thesis textFull thesis text - English Version


Abstract

NrCAM is a neuronal cell adhesion molecule of the L1 family of the immunoglobulin super-family. It plays a wide variety of roles in neural development including cell proliferation and differentiation, axon growth and guidance, synapse formation and the formation of the myelinated nerve structure. NrCAM over-expression was shown to be associated with cancer progression in diverse organs and with aggressive clinical phenotypes. NrCAM was also identified as an up-regulated gene in a tubular angiogenic in-vitro model of endothelial cells. We revealed that silencing NrCAM expression in human umbilical vein derived endothelial cells (HUVEC) was accompanied by inhibition of bFGF and VEGF induced cell proliferation, whereas ectopic over-expression of NrCAM resulted in a significant enhancement of pro-proliferative signals transduced by bFGF and VEGF. Furthermore, NrCAM formed stable complexes with the   FGFR-1 and VEGFR-2 tyrosine-kinase receptors. I demonstrated that over-expression of the full-length form of  NrCAM in PAE cells that also expressed  either  the recombinant  FGFR-1 or VEGFR-2, strongly potentiated bFGF-induced phosphorylation of FGFR-1 and VEGF-induced phosphorylation of VEGFR-2, respectively. Over-expression of full-length NrCAM was also accompanied with the up-regulation of the concentration levels of both FGFR-1 and VEGFR-2 in PAE cells. Silencing NrCAM expression in HUVEC also resulted in the inhibition of angiogenesis in in-vitro and in-vivo assays. These data strongly suggest that NrCAM functions as a pro-angiogenic factor that is required for the biological activity of the angiogenic factors bFGF and VEGF.

Class-3 semaphorins (sema3X) are secreted axon guidance factors. To determine if class-3 semaphorins are able to inhibit the development of glioblastoma multiforme (GBM) tumors, we expressed recombinant sema - 3A, 3B, 3D, 3E, 3F or 3G in U87MG glioblastoma cells. With the exception of sema3G, we revealed that expression of these semaphorins in U87MG cells strongly inhibited tumor development from subcutaneously implanted cells. We also have found that tumors that developed from cells expressing the most potent inhibitors, sema3A, sema3D, sema3E and sema3F, contained significantly reduced concentrations of blood vessels. Significant inhibition of tumor development was also observed following implantation of semaphorin expressing U87MG cells in the cortex of mouse brains. Sema3D and sema3E displayed the strongest inhibitory effects and indeed, their expression in U373MG or in U87MG glioblastoma cells implanted in the brains of mice prolonged the survival of the mice by more than two folds. Using CAM assay, we found that sema3D also functions as an inhibitor of angiogenesis in-vivo.