This proposal is based on the hypothesis that meschymal cells of the embroyonic kidney, expressing flk-1 and ELk - a form of tyrosine kinase receptors, are progenitors of endothelial cells responsible for the vascuolgenesis of the metanephros. these receptors under the influence of VEGF, a putative ligand for flk-1, undergo mitosis and migration; while the LERK, a putative ligand for ELK, is responsible for targeting and binding between anigoblasts to accomplish successful vasculogenesis of the kidney. The Principal Investigator and his collaborator, Dr Tom Daniel, believer that the proximal events involving EG:flk-1 interactions are responsible for vasculogenesis where here is coalescing of the capillaries, while the distal events involving LERK:ELK iinteractions are responsible for the angiogenesis by which the coalesced intrarenal islands of capillaries and connect to major blood vessels arising from dorsal aorta. To understand the processes of vascuolgenesis and angiogenesis, experiments are proposed utilizing multidisciplinary approaches under 3 specific aims.
the aim 1 relates to the isolation of the angioblasts from the embroyonic kidney. Embryonic kidneys, at day 11-12 of the gestation, from the mice cross between ROSA26 (b-galactosidase transgene) Immortomouse (H-2kb-tsA58) will be harvested. The cells will be dispersed with trypsin/EDTA and cultured under permissive (33oc) and non-permissive (37oc) conditions. After a certain number of defined passages, the cell lines will be screened for flk-1 and b- galactosidase activities. In addition, immunoflourescent microscopy, western blotting will be carried to ascertain the synthesis of receptor proteins. the lines positive for the flk-1 and b-galactosidase activities will be injected into the renal capsule of neonatal wild-type mice. The animals will be sacrificed after a week and kidneys processed b- galactosidase activity and expression of flk-1 by RT-PCR analyses.
In specific aim 2, flk-1 positive clones will be characterize under in vitro conditions for their capacity to response and proliferate under the influence of VEGF and LERk-2, degrade extracellular matrix, migrate and to form endothelial capillary-like structures by employing various methods such as, tyrosine phosphorylation assa, (3H)-thymidine incorporation, immunofluorescence of cytoskieletal organization, Boyden's chamber chemotaxis, zymography and matrigel capillary assembly assay.
The specific aim # 3 relates to the capacity of endothelial cells to form capillaries with functional disruption of flk- 1/ELK by their overexpression of soluble forms in cells infected withe XPress plasmid (pUHD13-1) in which the ELK or flk-1 gene segments are inserted downstream of a tetracycline responsive promoter. The Principal Investigator anticipates that the over-expression of secretory form flk-1 or ELK would block their intrinsic activities and would perform dominant-negative functions rather than stimulating phosphorylation and capillary-like tub formation.
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