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Abstract
Angiogenesis relies on the coordination of endothelial cells, smooth muscle cells and the vascular extracellular matrix around cells. Perlecan is an important heparan sulfate proteoglycan, which is widely expressed in vascular tissues and essentially involved in angiogenesis. Perlecan exhibits pro- and anti-angiogenic effects differently according to the cell line from which it was derived from and various structures especially its dependent glycosaminoglycans. The aim of the thesis was to generate and characterise monoclonal antibodies against specific domains of perlecan, and investigate their functions in modulating angiogenesis.
lmmunopurified human endothelial cell-derived perlecan decorated with heparin sulfate and recombinant perlecan domain V decorated with chondroitin sulfate were characterised by Enzyme linked immunosorbent assay, immunocytochemistry and Western blotting, and then used as antigens for hybridoma screening. There were 205 single clones generated by limiting dilution, while 174 clones were able to recognise endothelial perlecan. 48 clones with high viability and lg production were tested with intact perlecan and Hep Ill treated perlecan, while they showed different immunoreactive changes between two antigens. However, none of the clones reacted with recombinant perlecan domain I or domain V.
Of the newly generated monoclonal antibodies (mAbs), 5 mAbs showed potential to modulate the adhesion of endothelial cells and smooth muscle cells, especially 8C8 and 4F2 reduced the adhesion of both cell types. In addition, the 5 mAbs displayed strong immunoreactivity with perlecan derived from HCAECs and HVSMCs in Western blotting and were able to detect the perlecan expressed on two types of cells in immunocytochemistry. In spreading assay, all 5 mAbs were found to inhibit the HVSMC and HCAEC spreading to different degrees. Moreover, the 5 mAbs except 5G8 showed inhibitory effects in HCAEC migration, while only 8C8, 4F7 and 4F2 were able to reduce the migration of HVSMCs. These 5 mAbs could be useful to investigate the presence and structure of perlecan as primary antibodies, since they were able to recognise the perlecan protein core. They could also become potential tools to explain the functions of different domains of perlecan in angiogenesis and developed as immunotherapeutic drugs to mediate angiogenesis in clinical treatments.