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R. Longeras, J.–X. Ma; Angiogenic Inhibitor Kringle 5 And Three Deletion Mutants: Expression, Purification And Biological Activity . Invest. Ophthalmol. Vis. Sci. 2006;47(13):948.
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© ARVO (1962-2015); The Authors (2016-present)
There are two counter balancing systems that control angiogenesis: angiogenic stimulators (vascular endothelial growth factor) and angiogenic inhibitors: angiostatin, pigment epithelium derived factor (PEDF) and plasminogen Kringle 5. Kringle 5 is a naturally occurring angiogenic inhibitor also known to reduce vascular leakage. Studies have shown a hypoxia–generated shift in angiogenic balance leading to a decreased production of angiogenic inhibitors. We hypothesize that peptide domains of Kringle 5 are responsible for the anti–angiogenic activity or effects on vascular leakage induced via down–regulation of the VEGF expression. We sought to investigate the domain responsible for the biological activity by creating several Kringle 5 deletion mutants and assessing their activity and toxicity on cell culture.
Kringle 5 and three different Kringle 5 deletion mutants were purified out from the soluble fraction of E.coli. Bacteria were incubated at 25°C and IPTG–induced overnight. Each protein was treated separately. Subsequently, cell pellets were resuspended and treated with lysozyme for 1h. Soluble fractions were run through a nickel–affinity column and eluants dialyzed against 0.1X PBS for 4h. Purity of the above–purified peptides was assessed by SDS–page, identity was confirmed by Western blot and quantity was determined by Bradford method. Biological activity was controlled by subjecting Bovine Retina Capillary Endothelial Cells (BRCEC) to increasing amounts of each recombinant polypeptide. BCEC were isolated in our laboratory and subcultured on gelatin–coated wells in low glucose DMEM,1% FBS and 1% antibiotic/antifungal. Cell toxicity was assessed by MTT assay and controlled with Müller cells undergoing identical treatment.
Kringle 5 and deletion mutants 1, 2 and 3 were purified to homogeneity. The amount of recovered polypeptides varies from 5–10mg.L–1 for Kringle 5 to 1mg.L–1 for mutant 3. All four polypeptides exhibited strong specific antiproliferative proprieties on BRCEC whereas lacking nonspecific cytotoxicity towards Müller cells. Treatment with 100nM of Kringle 5 and all three mutants resulted in a similar proliferation decrease of ∼70%. ED50 for Kringle 5 and the three mutants was recorded to be 70nM.
Obtention to purity of three biologically active Kringle 5 deletion mutants opens the door to understanding specific angiogenic and inflammation mechanisms in the retina as well as the possibility to generate direct delivery routes for therapeutic treatments of pathologies of the eye.
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