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E. Im, A. Kazlauskas; Src Kinases and Pi3k Mediate Angiogenesis by Parallel Pathway . Invest. Ophthalmol. Vis. Sci. 2006;47(13):143.
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© ARVO (1962-2015); The Authors (2016-present)
Many intracellular signaling enzymes direct ocular angiogenic processes. Our previous studies showed that phosphoinositide 3 kinase (PI3K) promoted angiogenesis. In tumors, Src family kinases promoted angiogenesis by inducing vascular endothelial growth factor production. Moreover, other reports indicate that PI3K regulated angiogenesis in concert with Src family kinases by unknown mechanism. The purpose of this study is to investigate the mechanism of how Src and PI3K mediate angiogenic process.
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 platelet–derived growth factor ß 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 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. Akt phosphorylation was measured by Western blotting.
Consistent with our previous work, the receptor mutant capable of activating PI3K formed tubes. Receptors capable of activating both PI3K and Src formed tubes better than the PI3K receptor mutant. However, the receptor capable of activating Src alone induced tubes only when supplemented with PDGF exogenously. This suggested that Src was a weak driver of the tube response. As expected, PI3K was recruited to the PI3K receptor mutant, but not the Src receptor mutant. Furthermore, an inhibitor of PI3K (LY294002) blocked PI3K receptor mutant–induced tube, but did not affect Src receptor mutant driven tubes. Despite the apparent independence of the Src receptor mutant from PI3K, it promoted phosphorylation of Akt.
Our results revealed that in addition to PI3K, Src promoted tube formation. The two signaling enzymes converge on Akt. Our ongoing studies will test the mechanism of how Src activates Akt, which results in tube response and contributes to ocular angiogenesis in vivo.
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