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E. Im, A. Kazlauskas; Src Enhances in vitro Angiogenesis by Promoting Tube Formation and by Attenuating Rho-Dependent Regression. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1739.
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Angiogenesis is an essential pathological component for the manifestation of a wet form of age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity. Angiogenesis consists of a coordinated sequence of cellular events that are controlled by signaling enzymes. Similarly, vessel regression can be governed at the level of signal transduction pathways. Here we investigated the role of Src family kinases (SFKs) on the formation and regression of tubes.
We isolated primary bovine retinal endothelial cells (BRECs) and used them in an in vitro angiogenesis assay. These cells secrete PDGF, and by expressing the chimeric Î±/Î² platelet-derived growth factor receptor (PDGFR) in them, we established a system in which tube formation was dependent on PDGFR signaling. We also generated cells stably expressing a panel of chimeric PDGFR signaling mutants, which permitted us to assess the relationship between PI3K and Src. Recruitment of p85, an adaptor molecule of PI3K, to PDGFR was detected by immunoprecipitation followed by Western blotting. Phosphorylation of Akt and Erk was measured by Western blotting.
We report that SFKs promoted tube formation, however they were not as potent as phosphoinositide 3 kinase (PI3K). SFKs increased phosphorylation of Akt and PI3K inhibitor reduced tube formation of SFKs. In contrast to PI3K, SFKs potently attenuated the regression of tubes. The underlying mechanism appeared to involve SFK-dependent activation of Erk, which antagonized Rho. An inhibitor of Rho blocked tube regression, whereas Erk inhibitor promoted regression.
These studies reveal that SFKs promoted in vitro angiogenesis in two ways: 1) by promoting the formation of tubes, and 2) by antagonizing regression. Our ongoing studies will test the detailed mechanism of how SFKs promoted regression, which results in tube response and contributes to ocular angiogenesis in vivo.
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